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Sample records for label-free dna targets

  1. Switchable DNA interfaces for the highly sensitive detection of label-free DNA targets.

    Science.gov (United States)

    Rant, Ulrich; Arinaga, Kenji; Scherer, Simon; Pringsheim, Erika; Fujita, Shozo; Yokoyama, Naoki; Tornow, Marc; Abstreiter, Gerhard

    2007-10-30

    We report a method to detect label-free oligonucleotide targets. The conformation of surface-tethered probe nucleic acids is modulated by alternating electric fields, which cause the molecules to extend away from or fold onto the biased surface. Binding (hybridization) of targets to the single-stranded probes results in a pronounced enhancement of the layer-height modulation amplitude, monitored optically in real time. The method features an exceptional detection limit of <3 x 10(8) bound targets per cm(2) sensor area. Single base-pair mismatches in the sequences of DNA complements may readily be identified; moreover, binding kinetics and binding affinities can be determined with high accuracy. When driving the DNA to oscillate at frequencies in the kHz regime, distinct switching kinetics are revealed for single- and double-stranded DNA. Molecular dynamics are used to identify the binding state of molecules according to their characteristic kinetic fingerprints by using a chip-compatible detection format.

  2. A Fast, Sensitive and Label Free Electrochemical DNA Sensor

    International Nuclear Information System (INIS)

    Chen Yu; Elling; Lee Yokeling; Chong Serchoong

    2006-01-01

    A label free and sensitive DNA/RNA silicon based electrochemical microsensor array was developed by using thin film of the conducting polymer polypyrrole doped with an oligonucleotide probe. The electrochemical potential pulse amperometry technique was used for a biowarfare pathogen target DNA detection. The electrical potential assistanted DNA hybridisation method was applied. The sensor signal was increased by increasing the electrical potential assistanted DNA hybridisation time. It was possible to detect 0.34pmol and 0.072fmol of complementary oligonucleotide target in 0.1ml in seconds by using unpolished and polished gold electrode respectively. The probe preparation was also in seconds time, comparing indirect electrochemical DNA sensor, it has a fast sensor preparation as well as sensor response and label free advantages. The silicon microfabrication technique was used for this sensor array fabrication, which holds the potential to integrate with sensor electrical circuits. The conducting polymer polypyrrole was electrochemically deposited on each electrode respectively which has a possibility to dope the different DNA probe into the individual electrode to form a sensor array

  3. Label-free detection of DNA hybridization using carbon nanotube network field-effect transistors

    Science.gov (United States)

    Star, Alexander; Tu, Eugene; Niemann, Joseph; Gabriel, Jean-Christophe P.; Joiner, C. Steve; Valcke, Christian

    2006-01-01

    We report carbon nanotube network field-effect transistors (NTNFETs) that function as selective detectors of DNA immobilization and hybridization. NTNFETs with immobilized synthetic oligonucleotides have been shown to specifically recognize target DNA sequences, including H63D single-nucleotide polymorphism (SNP) discrimination in the HFE gene, responsible for hereditary hemochromatosis. The electronic responses of NTNFETs upon single-stranded DNA immobilization and subsequent DNA hybridization events were confirmed by using fluorescence-labeled oligonucleotides and then were further explored for label-free DNA detection at picomolar to micromolar concentrations. We have also observed a strong effect of DNA counterions on the electronic response, thus suggesting a charge-based mechanism of DNA detection using NTNFET devices. Implementation of label-free electronic detection assays using NTNFETs constitutes an important step toward low-cost, low-complexity, highly sensitive and accurate molecular diagnostics. hemochromatosis | SNP | biosensor

  4. Label-Free Potentiometry for Detecting DNA Hybridization Using Peptide Nucleic Acid and DNA Probes

    Directory of Open Access Journals (Sweden)

    Yuji Miyahara

    2013-02-01

    Full Text Available Peptide nucleic acid (PNA has outstanding affinity over DNA for complementary nucleic acid sequences by forming a PNA-DNA heterodimer upon hybridization via Watson-Crick base-pairing. To verify whether PNA probes on an electrode surface enhance sensitivity for potentiometric DNA detection or not, we conducted a comparative study on the hybridization of PNA and DNA probes on the surface of a 10-channel gold electrodes microarray. Changes in the charge density as a result of hybridization at the solution/electrode interface on the self-assembled monolayer (SAM-formed microelectrodes were directly transformed into potentiometric signals using a high input impedance electrometer. The charge readout allows label-free, reagent-less, and multi-parallel detection of target oligonucleotides without any optical assistance. The differences in the probe lengths between 15- to 22-mer dramatically influenced on the sensitivity of the PNA and DNA sensors. Molecular type of the capturing probe did not affect the degree of potential shift. Theoretical model for charged rod-like duplex using the Gouy-Chapman equation indicates the dominant effect of electrostatic attractive forces between anionic DNA and underlying electrode at the electrolyte/electrode interface in the potentiometry.

  5. Label-Free Potentiometry for Detecting DNA Hybridization Using Peptide Nucleic Acid and DNA Probes

    Science.gov (United States)

    Goda, Tatsuro; Singi, Ankit Balram; Maeda, Yasuhiro; Matsumoto, Akira; Torimura, Masaki; Aoki, Hiroshi; Miyahara, Yuji

    2013-01-01

    Peptide nucleic acid (PNA) has outstanding affinity over DNA for complementary nucleic acid sequences by forming a PNA-DNA heterodimer upon hybridization via Watson-Crick base-pairing. To verify whether PNA probes on an electrode surface enhance sensitivity for potentiometric DNA detection or not, we conducted a comparative study on the hybridization of PNA and DNA probes on the surface of a 10-channel gold electrodes microarray. Changes in the charge density as a result of hybridization at the solution/electrode interface on the self-assembled monolayer (SAM)-formed microelectrodes were directly transformed into potentiometric signals using a high input impedance electrometer. The charge readout allows label-free, reagent-less, and multi-parallel detection of target oligonucleotides without any optical assistance. The differences in the probe lengths between 15- to 22-mer dramatically influenced on the sensitivity of the PNA and DNA sensors. Molecular type of the capturing probe did not affect the degree of potential shift. Theoretical model for charged rod-like duplex using the Gouy-Chapman equation indicates the dominant effect of electrostatic attractive forces between anionic DNA and underlying electrode at the electrolyte/electrode interface in the potentiometry. PMID:23435052

  6. Biosensor for label-free DNA quantification based on functionalized LPGs.

    Science.gov (United States)

    Gonçalves, Helena M R; Moreira, Luis; Pereira, Leonor; Jorge, Pedro; Gouveia, Carlos; Martins-Lopes, Paula; Fernandes, José R A

    2016-10-15

    A label-free fiber optic biosensor based on a long period grating (LPG) and a basic optical interrogation scheme using off the shelf components is used for the detection of in-situ DNA hybridization. A new methodology is proposed for the determination of the spectral position of the LPG mode resonance. The experimental limit of detection obtained for the DNA was 62±2nM and the limit of quantification was 209±7nM. The sample specificity was experimentally demonstrated using DNA targets with different base mismatches relatively to the probe and was found that the system has a single base mismatch selectivity. Copyright © 2015 Elsevier B.V. All rights reserved.

  7. Tyramine Hydrochloride Based Label-Free System for Operating Various DNA Logic Gates and a DNA Caliper for Base Number Measurements.

    Science.gov (United States)

    Fan, Daoqing; Zhu, Xiaoqing; Dong, Shaojun; Wang, Erkang

    2017-07-05

    DNA is believed to be a promising candidate for molecular logic computation, and the fluorogenic/colorimetric substrates of G-quadruplex DNAzyme (G4zyme) are broadly used as label-free output reporters of DNA logic circuits. Herein, for the first time, tyramine-HCl (a fluorogenic substrate of G4zyme) is applied to DNA logic computation and a series of label-free DNA-input logic gates, including elementary AND, OR, and INHIBIT logic gates, as well as a two to one encoder, are constructed. Furthermore, a DNA caliper that can measure the base number of target DNA as low as three bases is also fabricated. This DNA caliper can also perform concatenated AND-AND logic computation to fulfil the requirements of sophisticated logic computing. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Label-free integrative pharmacology on-target of drugs at the β2-adrenergic receptor

    Science.gov (United States)

    Ferrie, Ann M.; Sun, Haiyan; Fang, Ye

    2011-07-01

    We describe a label-free integrative pharmacology on-target (iPOT) method to assess the pharmacology of drugs at the β2-adrenergic receptor. This method combines dynamic mass redistribution (DMR) assays using an array of probe molecule-hijacked cells with similarity analysis. The whole cell DMR assays track cell system-based, ligand-directed, and kinetics-dependent biased activities of the drugs, and translates their on-target pharmacology into numerical descriptors which are subject to similarity analysis. We demonstrate that the approach establishes an effective link between the label-free pharmacology and in vivo therapeutic indications of drugs.

  9. Improving Probe Immobilization for Label-Free Capacitive Detection of DNA Hybridization on Microfabricated Gold Electrodes

    Directory of Open Access Journals (Sweden)

    Sandro Carrara

    2008-02-01

    Full Text Available Alternative approaches to labeled optical detection for DNA arrays are actively investigated for low-cost point-of-care applications. In this domain, label-free capacitive detection is one of the most intensely studied techniques. It is based on the idea to detect the Helmholtz ion layer displacements when molecular recognition occurs at the electrodes/solution interface. The sensing layer is usually prepared by using thiols terminated DNA single-strength oligonucleotide probes on top of the sensor electrodes. However, published data shows evident time drift, which greatly complicates signal conditioning and processing and ultimately increases the uncertainty in DNA recognition sensing. The aim of this work is to show that newly developed ethylene-glycol functionalized alkanethiols greatly reduce time drift, thereby significantly improving capacitance based label-free detection of DNA.

  10. Label-free detection of biomolecular interaction — DNA — Antimicrobial peptide binding

    DEFF Research Database (Denmark)

    Fojan, Peter; Jensen, Kasper Risgaard; Gurevich, Leonid

    2011-01-01

    the molecule. In particular, surface plasmon resonance (SPR) sensors have been already demonstrated suitable for food-safety control, label-free screening for various disease markers in bodily fluids, as well as for real-time continuous monitoring of drug levels in intensive care environment. We envisage...... of plasmon based biosensors to the study of the interaction of Antimicrobial peptide IL4 and DNA. Our results indicate high affinity binding between IL4 and DNA thereby preventing DNA replication and eventually killing the affected cell. We speculate that this is common for a large class of Antimicrobial...

  11. Electrochemical direct immobilization of DNA sequences for label-free herpes virus detection

    Science.gov (United States)

    Tam, Phuong Dinh; Trung, Tran; Tuan, Mai Anh; Chien, Nguyen Duc

    2009-09-01

    DNA sequences/bio-macromolecules of herpes virus (5'-AT CAC CGA CCC GGA GAG GGA C-3') were directly immobilized into polypyrrole matrix by using the cyclic voltammetry method, and grafted onto arrays of interdigitated platinum microelectrodes. The morphology surface of the obtained PPy/DNA of herpes virus composite films was investigated by a FESEM Hitachi-S 4800. Fourier transform infrared spectroscopy (FTIR) was used to characterize the PPy/DNA film and to study the specific interactions that may exist between DNA biomacromolecules and PPy chains. Attempts are made to use these PPy/DNA composite films for label-free herpes virus detection revealed a response time of 60 s in solutions containing as low as 2 nM DNA concentration, and self life of six months when immerged in double distilled water and kept refrigerated.

  12. Electrochemical direct immobilization of DNA sequences for label-free herpes virus detection

    International Nuclear Information System (INIS)

    Phuong Dinh Tam; Mai Anh Tuan; Tran Trung; Nguyen Duc Chien

    2009-01-01

    DNA sequences/bio-macromolecules of herpes virus (5'-AT CAC CGA CCC GGA GAG GGA C-3') were directly immobilized into polypyrrole matrix by using the cyclic voltammetry method, and grafted onto arrays of interdigitated platinum microelectrodes. The morphology surface of the obtained PPy/DNA of herpes virus composite films was investigated by a FESEM Hitachi-S 4800. Fourier transform infrared spectroscopy (FTIR) was used to characterize the PPy/DNA film and to study the specific interactions that may exist between DNA biomacromolecules and PPy chains. Attempts are made to use these PPy/DNA composite films for label-free herpes virus detection revealed a response time of 60 s in solutions containing as low as 2 nM DNA concentration, and self life of six months when emerged in double distilled water and kept refrigerated.

  13. Electrochemical direct immobilization of DNA sequences for label-free herpes virus detection

    Energy Technology Data Exchange (ETDEWEB)

    Phuong Dinh Tam; Mai Anh Tuan [International Training Institute for Materials Science (Viet Nam); Tran Trung [Department of Electrochemistry, Hung-Yen University of Technology and Education (Viet Nam); Nguyen Duc Chien [Institute of Engineering Physics, Hanoi University of Technology, 1 Dai Co Viet Road, Hanoi (Viet Nam)], E-mail: tr_trunghut@yahoo.com

    2009-09-01

    DNA sequences/bio-macromolecules of herpes virus (5'-AT CAC CGA CCC GGA GAG GGA C-3') were directly immobilized into polypyrrole matrix by using the cyclic voltammetry method, and grafted onto arrays of interdigitated platinum microelectrodes. The morphology surface of the obtained PPy/DNA of herpes virus composite films was investigated by a FESEM Hitachi-S 4800. Fourier transform infrared spectroscopy (FTIR) was used to characterize the PPy/DNA film and to study the specific interactions that may exist between DNA biomacromolecules and PPy chains. Attempts are made to use these PPy/DNA composite films for label-free herpes virus detection revealed a response time of 60 s in solutions containing as low as 2 nM DNA concentration, and self life of six months when emerged in double distilled water and kept refrigerated.

  14. Asynchronous Magnetic Bead Rotation (AMBR Microviscometer for Label-Free DNA Analysis

    Directory of Open Access Journals (Sweden)

    Yunzi Li

    2014-03-01

    Full Text Available We have developed a label-free viscosity-based DNA detection system, using paramagnetic beads as an asynchronous magnetic bead rotation (AMBR microviscometer. We have demonstrated experimentally that the bead rotation period is linearly proportional to the viscosity of a DNA solution surrounding the paramagnetic bead, as expected theoretically. Simple optical measurement of asynchronous microbead motion determines solution viscosity precisely in microscale volumes, thus allowing an estimate of DNA concentration or average fragment length. The response of the AMBR microviscometer yields reproducible measurement of DNA solutions, enzymatic digestion reactions, and PCR systems at template concentrations across a 5000-fold range. The results demonstrate the feasibility of viscosity-based DNA detection using AMBR in microscale aqueous volumes.

  15. Label-free detection of DNA hybridization and single point mutations in a nano-gap biosensor

    International Nuclear Information System (INIS)

    Zaffino, R L; Mir, M; Samitier, J

    2014-01-01

    We describe a conductance-based biosensor that exploits DNA-mediated long-range electron transport for the label-free and direct electrical detection of DNA hybridization. This biosensor platform comprises an array of vertical nano-gap biosensors made of gold and fabricated through standard photolithography combined with focused ion beam lithography. The nano-gap walls are covalently modified with short, anti-symmetric thiolated DNA probes, which are terminated by 19 bases complementary to both the ends of a target DNA strand. The nano-gaps are separated by a distance of 50nm, which was adjusted to fit the length of the DNA target plus the DNA probes. The hybridization of the target DNA closes the gap circuit in a switch on/off fashion, in such a way that it is readily detected by an increase in the current after nano-gap closure. The nano-biosensor shows high specificity in the discrimination of base-pair mismatching and does not require signal indicators or enhancing molecules. The design of the biosensor platform is applicable for multiplexed detection in a straightforward manner. The platform is well-suited to mass production, point-of-care diagnostics, and wide-scale DNA analysis applications. (paper)

  16. Electrochemical label-free and sensitive nanobiosensing of DNA hybridization by graphene oxide modified pencil graphite electrode.

    Science.gov (United States)

    Ahour, F; Shamsi, A

    2017-09-01

    Based on the strong interaction between single-stranded DNA (ss-DNA) and graphene material, we have constructed a novel label-free electrochemical biosensor for rapid and facile detection of short sequences ss-DNA molecules related to hepatitis C virus 1a using graphene oxide modified pencil graphite electrode. The sensing mechanism is based on the superior adsorption of single-stranded DNA to GO over double stranded DNA (ds-DNA). The intrinsic guanine oxidation signal measured by differential pulse voltammetry (DPV) has been used for duplex DNA formation detection. The probe ss-DNA adsorbs onto the surface of GO via the π- π* stacking interactions leading to a strong background guanine oxidation signal. In the presence of complementary target, formation of helix which has weak binding ability to GO induced ds-DNA to release from the electrode surface and significant variation in differential pulse voltammetric response of guanine bases. The results indicated that the oxidation peak current was proportional to the concentration of complementary strand in the range of 0.1 nM-0.5 μM with a detection limit of 4.3 × 10 -11  M. The simple fabricated electrochemical biosensor has high sensitivity, good selectivity, and could be applied as a new platform for a range of target molecules in future. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. Quenching the chemiluminescence of acridinium ester by graphene oxide for label-free and homogeneous DNA detection.

    Science.gov (United States)

    He, Yi; Huang, Guangming; Cui, Hua

    2013-11-13

    It was found that graphene oxide (GO) could effectively quench the chemiluminescence (CL) emission from a acridinium ester (AE)-hydrogen peroxide system. By taking advantage of this quenching effect, as a proof of concept, a label-free and homogeneous DNA assay was developed for the detection of Mycobacterium tuberculosis DNA. In the absence of target DNA, both probe DNA and AE were absorbed on the surface of GO, producing a weak CL emission owing to the CL quenching effect of GO. However, in the presence of target DNA, a double-stranded structure of DNA was generated, leading to the release of the oligonucleotide from the GO surface. AE favors binding with double-stranded DNA, which will be released from the GO surface; thus, the quenching effect of GO will be no longer effective and a strong CL signal can be observed. This assay can detect M. tuberculosis DNA with a detection limit of 0.65 nM. This sensitivity is lower than that of previously reported electrochemical detection.

  18. Label-Free Ag+ Detection by Enhancing DNA Sensitized Tb3+ Luminescence

    Directory of Open Access Journals (Sweden)

    Kimberly Kleinke

    2016-08-01

    Full Text Available In this work, the effect of Ag+ on DNA sensitized Tb3+ luminescence was studied initially using the Ag+-specific RNA-cleaving DNAzyme, Ag10c. While we expected to observe luminescence quenching by Ag+, a significant enhancement was produced. Based on this observation, simple DNA oligonucleotide homopolymers were used with systematically varied sequence and length. We discovered that both poly-G and poly-T DNA have a significant emission enhancement by Ag+, while the absolute intensity is stronger with the poly-G DNA, indicating that a G-quadruplex DNA is not required for this enhancement. Using the optimized length of the G7 DNA (an oligo constituted with seven guanines, Ag+ was measured with a detection limit of 57.6 nM. The signaling kinetics, G7 DNA conformation, and the binding affinity of Tb3+ to the DNA in the presence or absence of Ag+ are also studied to reveal the mechanism of emission enhancement. This observation is useful not only for label-free detection of Ag+, but also interesting for the rational design of new biosensors using Tb3+ luminescence.

  19. Label-free detection of DNA hybridization using transistors based on CVD grown graphene.

    Science.gov (United States)

    Chen, Tzu-Yin; Loan, Phan Thi Kim; Hsu, Chang-Lung; Lee, Yi-Hsien; Tse-Wei Wang, Jacob; Wei, Kung-Hwa; Lin, Cheng-Te; Li, Lain-Jong

    2013-03-15

    The high transconductance and low noise of graphene-based field-effect transistors based on large-area monolayer graphene produced by chemical vapor deposition are used for label-free electrical detection of DNA hybridization. The gate materials, buffer concentration and surface condition of graphene have been optimized to achieve the DNA detection sensitivity as low as 1 pM (10(-12) M), which is more sensitive than the existing report based on few-layer graphene. The graphene films obtained using conventional PMMA-assisted transfer technique exhibits PMMA residues, which degrade the sensing performance of graphene. We have demonstrated that the sensing performance of the graphene samples prepared by gold-transfer is largely enhanced (by 125%). Copyright © 2012 Elsevier B.V. All rights reserved.

  20. Gold surface supported spherical liposome-gold nano-particle nano-composite for label free DNA sensing.

    Science.gov (United States)

    Bhuvana, M; Narayanan, J Shankara; Dharuman, V; Teng, W; Hahn, J H; Jayakumar, K

    2013-03-15

    Immobilization of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) liposome-gold nano-particle (DOPE-AuNP) nano-composite covalently on 3-mercaptopropionic acid (MPA) on gold surface is demonstrated for the first time for electrochemical label free DNA sensing. Spherical nature of the DOPE on the MPA monolayer is confirmed by the appearance of sigmoidal voltammetric profile, characteristic behavior of linear diffusion, for the MPA-DOPE in presence of [Fe(CN)(6)](3-/4-) and [Ru(NH(3))(6)](3+) redox probes. The DOPE liposome vesicle fusion is prevented by electroless deposition of AuNP on the hydrophilic amine head groups of the DOPE. Immobilization of single stranded DNA (ssDNA) is made via simple gold-thiol linkage for DNA hybridization sensing in the presence of [Fe(CN)(6)](3-/4-). The sensor discriminates the hybridized (complementary target hybridized), un-hybridized (non-complementary target hybridized) and single base mismatch target hybridized surfaces sensitively and selectively without signal amplification. The lowest target DNA concentration detected is 0.1×10(-12)M. Cyclic voltammetry (CV), electrochemical impedance (EIS), differential pulse voltammetry (DPV) and quartz crystal microbalance (QCM) techniques are used for DNA sensing on DOPE-AuNP nano-composite. Transmission Electron Microscopy (TEM), Fourier Transform Infrared Spectroscopy (FTIR), Atomic Force Microscopy (AFM), Dynamic Light Scattering (DLS) and Ultraviolet-Visible (UV) spectroscopic techniques are used to understand the interactions between the DOPE, AuNP and ssDNA. The results indicate the presence of an intact and well defined spherical DOPE-AuNP nano-composite on the gold surface. The method could be applied for fabrication of the surface based liposome-AuNP-DNA composite for cell transfection studies at reduced reagents and costs. Copyright © 2012 Elsevier B.V. All rights reserved.

  1. Organophosphonate-based PNA-functionalization of silicon nanowires for label-free DNA detection.

    Science.gov (United States)

    Cattani-Scholz, Anna; Pedone, Daniel; Dubey, Manish; Neppl, Stefan; Nickel, Bert; Feulner, Peter; Schwartz, Jeffrey; Abstreiter, Gerhard; Tornow, Marc

    2008-08-01

    We investigated hydroxyalkylphosphonate monolayers as a novel platform for the biofunctionalization of silicon-based field effect sensor devices. This included a detailed study of the thin film properties of organophosphonate films on Si substrates using several surface analysis techniques, including AFM, ellipsometry, contact angle, X-ray photoelectron spectroscopy (XPS), X-ray reflectivity, and current-voltage characteristics in electrolyte solution. Our results indicate the formation of a dense monolayer on the native silicon oxide that has excellent passivation properties. The monolayer was biofunctionalized with 12 mer peptide nucleic acid (PNA) receptor molecules in a two-step procedure using the heterobifunctional linker, 3-maleimidopropionic-acid-N-hydroxysuccinimidester. Successful surface modification with the probe PNA was verified by XPS and contact angle measurements, and hybridization with DNA was determined by fluorescence measurements. Finally, the PNA functionalization protocol was translated to 2 microm long, 100 nm wide Si nanowire field effect devices, which were successfully used for label-free DNA/PNA hybridization detection.

  2. Enzyme-free and label-free ultrasensitive electrochemical detection of DNA and adenosine triphosphate by dendritic DNA concatamer-based signal amplification.

    Science.gov (United States)

    Liu, Shufeng; Lin, Ying; Liu, Tao; Cheng, Chuanbin; Wei, Wenji; Wang, Li; Li, Feng

    2014-06-15

    Hybridization chain reaction (HCR) strategy has been well developed for the fabrication of various biosensing platforms for signal amplification. Herein, a novel enzyme-free and label-free ultrasensitive electrochemical DNA biosensing platform for the detection of target DNA and adenosine triphosphate (ATP) was firstly proposed, in which three auxiliary DNA probes were ingeniously designed to construct the dendritic DNA concatamer via HCR strategy and used as hexaammineruthenium(III) chloride (RuHex) carrier for signal amplification. With the developed dendritic DNA concatamer-based signal amplification strategy, the DNA biosensor could achieve an ultrasensitive electrochemical detection of DNA and ATP with a superior detection limit as low as 5 aM and 20 fM, respectively, and also demonstrate a high selectivity for DNA and ATP detection. The currently proposed dendritic DNA concatamer opens a promising direction to construct ultrasensitive DNA biosensing platform for biomolecular detection in bioanalysis and clinical biomedicine, which offers the distinct advantages of simplicity and cost efficiency owing to no need of any kind of enzyme, chemical modification or labeling. Copyright © 2014 Elsevier B.V. All rights reserved.

  3. Self-reference and random sampling approach for label-free identification of DNA composition using plasmonic nanomaterials.

    Science.gov (United States)

    Freeman, Lindsay M; Pang, Lin; Fainman, Yeshaiahu

    2018-05-09

    The analysis of DNA has led to revolutionary advancements in the fields of medical diagnostics, genomics, prenatal screening, and forensic science, with the global DNA testing market expected to reach revenues of USD 10.04 billion per year by 2020. However, the current methods for DNA analysis remain dependent on the necessity for fluorophores or conjugated proteins, leading to high costs associated with consumable materials and manual labor. Here, we demonstrate a potential label-free DNA composition detection method using surface-enhanced Raman spectroscopy (SERS) in which we identify the composition of cytosine and adenine within single strands of DNA. This approach depends on the fact that there is one phosphate backbone per nucleotide, which we use as a reference to compensate for systematic measurement variations. We utilize plasmonic nanomaterials with random Raman sampling to perform label-free detection of the nucleotide composition within DNA strands, generating a calibration curve from standard samples of DNA and demonstrating the capability of resolving the nucleotide composition. The work represents an innovative way for detection of the DNA composition within DNA strands without the necessity of attached labels, offering a highly sensitive and reproducible method that factors in random sampling to minimize error.

  4. A graphene oxide-based sensing platform for the label-free assay of DNA sequence and exonuclease activity via long range resonance energy transfer.

    Science.gov (United States)

    Jiang, Yixuan; Tian, Jianniao; Chen, Sheng; Zhao, Yanchun; Wang, Yuan; Zhao, Shulin

    2013-07-01

    Graphene oxide (GO) was introduced as an efficient quencher for label-free and sensitive detection of DNA. Probe DNA (pDNA) was mixed with ethidium bromide (EB) and graphene oxide (GO). The interaction between EB and GO led to the fluorescent quenching. Upon the recognition of the target, EB was intercalated into duplex DNA and kept away from GO, which significantly hindered the long range resonance energy transfer (LrRET) from EB to GO and, thus, increased the fluorescence of EB. The changes in fluorescent intensity produced a novel method for sensitivity, and specificity detection of the target. Based on the structure-switching of aptamers, this strategy could be conveniently extended for detection of other biomolecules, which had been demonstrated by the detection of exonuclease activity.

  5. New Detection Modality for Label-Free Quantification of DNA in Biological Samples via Superparamagnetic Bead Aggregation

    Science.gov (United States)

    Leslie, Daniel C.; Li, Jingyi; Strachan, Briony C.; Begley, Matthew R.; Finkler, David; Bazydlo, Lindsay L.; Barker, N. Scott; Haverstick, Doris; Utz, Marcel; Landers, James P.

    2012-01-01

    Combining DNA and superparamagnetic beads in a rotating magnetic field produces multiparticle aggregates that are visually striking, and enables label-free optical detection and quantification of DNA at levels in the picogram per microliter range. DNA in biological samples can be quantified directly by simple analysis of optical images of microfluidic wells placed on a magnetic stirrer without DNA purification. Aggregation results from DNA/bead interactions driven either by the presence of a chaotrope (a nonspecific trigger for aggregation) or by hybridization with oligonucleotides on functionalized beads (sequence-specific). This paper demonstrates quantification of DNA with sensitivity comparable to that of the best currently available fluorometric assays. The robustness and sensitivity of the method enable a wide range of applications, illustrated here by counting eukaryotic cells. Using widely available and inexpensive benchtop hardware, the approach provides a highly accessible low-tech microscale alternative to more expensive DNA detection and cell counting techniques. PMID:22423674

  6. Hall effect biosensors with ultraclean graphene film for improved sensitivity of label-free DNA detection

    KAUST Repository

    Loan, Phan Thi Kim

    2017-07-19

    The quality of graphene strongly affects the performance of graphene-based biosensors which are highly demanded for the sensitive and selective detection of biomolecules, such as DNA. This work reported a novel transfer process for preparing a residue-free graphene film using a thin gold supporting layer. A Hall effect device made of this gold-transferred graphene was demonstrated to significantly enhance the sensitivity (≈ 5 times) for hybridization detection, with a linear detection range of 1 pM – 100nM for DNA target. Our findings provide an efficient method to boost the sensitivity of graphene-based biosensors for DNA recognition.

  7. Label-Free Fluorescent Detection of Trypsin Activity Based on DNA-Stabilized Silver Nanocluster-Peptide Conjugates

    Directory of Open Access Journals (Sweden)

    Cai-Xia Zhuo

    2016-11-01

    Full Text Available Trypsin is important during the regulation of pancreatic exocrine function. The detection of trypsin activity is currently limited because of the need for the substrate to be labeled with a fluorescent tag. A label-free fluorescent method has been developed to monitor trypsin activity. The designed peptide probe consists of six arginine molecules and a cysteine terminus and can be conjugated to DNA-stabilized silver nanoclusters (DNA-AgNCs by Ag-S bonding to enhance fluorescence. The peptide probe can also be adsorbed to the surface of graphene oxide (GO, thus resulting in the fluorescence quenching of DNA-AgNCs-peptide conjugate because of Förster resonance energy transfer. Once trypsin had degraded the peptide probe into amino acid residues, the DNA-AgNCs were released from the surface of GO, and the enhanced fluorescence of DNA-AgNCs was restored. Trypsin can be determined with a linear range of 0.0–50.0 ng/mL with a concentration as low as 1 ng/mL. This label-free method is simple and sensitive and has been successfully used for the determination of trypsin in serum. The method can also be modified to detect other proteases.

  8. Rapid and label-free electrochemical DNA biosensor for detecting hepatitis A virus.

    Science.gov (United States)

    Manzano, Marisa; Viezzi, Sara; Mazerat, Sandra; Marks, Robert S; Vidic, Jasmina

    2018-02-15

    Diagnostic systems that can deliver highly specific and sensitive detection of hepatitis A virus (HAV) in food and water are of particular interest in many fields including food safety, biosecurity and control of outbreaks. Our aim was the development of an electrochemical method based on DNA hybridization to detect HAV. A ssDNA probe specific for HAV (capture probe) was designed and tested on DNAs from various viral and bacterial samples using Nested-Reverse Transcription Polymerase Chain Reaction (nRT-PCR). To develop the electrochemical device, a disposable gold electrode was functionalized with the specific capture probe and tested on complementary ssDNA and on HAV cDNA. The DNA hybridization on the electrode was measured through the monitoring of the oxidative peak potential of the indicator tripropylamine by cyclic voltammetry. To prevent non-specific binding the gold surface was treated with 3% BSA before detection. High resolution atomic force microscopy (AFM) confirmed the efficiency of electrode functionalization and on-electrode hybridization. The proposed device showed a limit of detection of 0.65pM for the complementary ssDNA and 6.94fg/µL for viral cDNA. For a comparison, nRT-PCR quantified the target HAV cDNA with a limit of detection of 6.4fg/µL. The DNA-sensor developed can be adapted to a portable format to be adopted as an easy-to- use and low cost method for screening HAV in contaminated food and water. In addition, it can be useful for rapid control of HAV infections as it takes only a few minutes to provide the results. Copyright © 2017. Published by Elsevier B.V.

  9. Hall effect biosensors with ultraclean graphene film for improved sensitivity of label-free DNA detection.

    Science.gov (United States)

    Loan, Phan Thi Kim; Wu, Dongqin; Ye, Chen; Li, Xiaoqing; Tra, Vu Thanh; Wei, Qiuping; Fu, Li; Yu, Aimin; Li, Lain-Jong; Lin, Cheng-Te

    2018-01-15

    The quality of graphene strongly affects the performance of graphene-based biosensors which are highly demanded for the sensitive and selective detection of biomolecules, such as DNA. This work reported a novel transfer process for preparing a residue-free graphene film using a thin gold supporting layer. A Hall effect device made of this gold-transferred graphene was demonstrated to significantly enhance the sensitivity (≈ 5 times) for hybridization detection, with a linear detection range of 1pM to 100nM for DNA target. Our findings provide an efficient method to boost the sensitivity of graphene-based biosensors for DNA recognition. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. Graphene electrode modified with electrochemically reduced graphene oxide for label-free DNA detection.

    Science.gov (United States)

    Li, Bing; Pan, Genhua; Avent, Neil D; Lowry, Roy B; Madgett, Tracey E; Waines, Paul L

    2015-10-15

    A novel printed graphene electrode modified with electrochemically reduced graphene oxide was developed for the detection of a specific oligonucleotide sequence. The graphene oxide was immobilized onto the surface of a graphene electrode via π-π bonds and electrochemical reduction of graphene oxide was achieved by cyclic voltammetry. A much higher redox current was observed from the reduced graphene oxide-graphene double-layer electrode, a 42% and 36.7% increase, respectively, in comparison with that of a bare printed graphene or reduced graphene oxide electrode. The good electron transfer activity is attributed to a combination of the large number of electroactive sites in reduced graphene oxide and the high conductivity nature of graphene. The probe ssDNA was further immobilized onto the surface of the reduced graphene oxide-graphene double-layer electrode via π-π bonds and then hybridized with its target cDNA. The change of peak current due to the hybridized dsDNA could be used for quantitative sensing of DNA concentration. It has been demonstrated that a linear range from 10(-7)M to 10(-12)M is achievable for the detection of human immunodeficiency virus 1 gene with a detection limit of 1.58 × 10(-13)M as determined by three times standard deviation of zero DNA concentration. Copyright © 2015 Elsevier B.V. All rights reserved.

  11. Label-Free Detection of Sequence-Specific DNA Based on Fluorescent Silver Nanoclusters-Assisted Surface Plasmon-Enhanced Energy Transfer.

    Science.gov (United States)

    Ma, Jin-Liang; Yin, Bin-Cheng; Le, Huynh-Nhu; Ye, Bang-Ce

    2015-06-17

    We have developed a label-free method for sequence-specific DNA detection based on surface plasmon enhanced energy transfer (SPEET) process between fluorescent DNA/AgNC string and gold nanoparticles (AuNPs). DNA/AgNC string, prepared by a single-stranded DNA template encoded two emitter-nucleation sequences at its termini and an oligo spacer in the middle, was rationally designed to produce bright fluorescence emission. The proposed method takes advantage of two strategies. The first one is the difference in binding properties of single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) toward AuNPs. The second one is SPEET process between fluorescent DNA/AgNC string and AuNPs, in which fluorescent DNA/AgNC string can be spontaneously adsorbed onto the surface of AuNPs and correspondingly AuNPs serve as "nanoquencher" to quench the fluorescence of DNA/AgNC string. In the presence of target DNA, the sensing probe hybridized with target DNA to form duplex DNA, leading to a salt-induced AuNP aggregation and subsequently weakened SPEET process between fluorescent DNA/AgNC string and AuNPs. A red-to-blue color change of AuNPs and a concomitant fluorescence increase were clearly observed in the sensing system, which had a concentration dependent manner with specific DNA. The proposed method achieved a detection limit of ∼2.5 nM, offering the following merits of simple design, convenient operation, and low experimental cost because of no chemical modification, organic dye, enzymatic reaction, or separation procedure involved.

  12. The field effect transistor DNA biosensor based on ITO nanowires in label-free hepatitis B virus detecting compatible with CMOS technology.

    Science.gov (United States)

    Shariati, Mohsen

    2018-05-15

    In this paper the field-effect transistor DNA biosensor for detecting hepatitis B virus (HBV) based on indium tin oxide nanowires (ITO NWs) in label free approach has been fabricated. Because of ITO nanowires intensive conductance and functional modified surface, the probe immobilization and target hybridization were increased strongly. The high resolution transmission electron microscopy (HRTEM) measurement showed that ITO nanowires were crystalline and less than 50nm in diameter. The single-stranded hepatitis B virus DNA (SS-DNA) was immobilized as probe on the Au-modified nanowires. The DNA targets were measured in a linear concentration range from 1fM to 10µM. The detection limit of the DNA biosensor was about 1fM. The time of the hybridization process for defined single strand was 90min. The switching ratio of the biosensor between "on" and "off" state was ~ 1.1 × 10 5 . For sensing the specificity of the biosensor, non-complementary, mismatch and complementary DNA oligonucleotide sequences were clearly discriminated. The HBV biosensor confirmed the highly satisfied specificity for differentiating complementary sequences from non-complementary and the mismatch oligonucleotides. The response time of the DNA sensor was 37s with a high reproducibility. The stability and repeatability of the DNA biosensor showed that the peak current of the biosensor retained 98% and 96% of its initial response for measurements after three and five weeks, respectively. Copyright © 2018 Elsevier B.V. All rights reserved.

  13. Mapping in vivo target interaction profiles of covalent inhibitors using chemical proteomics with label-free quantification.

    Science.gov (United States)

    van Rooden, Eva J; Florea, Bogdan I; Deng, Hui; Baggelaar, Marc P; van Esbroeck, Annelot C M; Zhou, Juan; Overkleeft, Herman S; van der Stelt, Mario

    2018-04-01

    Activity-based protein profiling (ABPP) has emerged as a valuable chemical proteomics method to guide the therapeutic development of covalent drugs by assessing their on-target engagement and off-target activity. We recently used ABPP to determine the serine hydrolase interaction landscape of the experimental drug BIA 10-2474, thereby providing a potential explanation for the adverse side effects observed with this compound. ABPP allows mapping of protein interaction landscapes of inhibitors in cells, tissues and animal models. Whereas our previous protocol described quantification of proteasome activity using stable-isotope labeling, this protocol describes the procedures for identifying the in vivo selectivity profile of covalent inhibitors with label-free quantitative proteomics. The optimization of our protocol for label-free quantification methods results in high proteome coverage and allows the comparison of multiple biological samples. We demonstrate our protocol by assessing the protein interaction landscape of the diacylglycerol lipase inhibitor DH376 in mouse brain, liver, kidney and testes. The stages of the protocol include tissue lysis, probe incubation, target enrichment, sample preparation, liquid chromatography-mass spectrometry (LC-MS) measurement, data processing and analysis. This approach can be used to study target engagement in a native proteome and to identify potential off targets for the inhibitor under investigation. The entire protocol takes at least 4 d, depending on the number of samples.

  14. Label-free DNA quantification via a 'pipette, aggregate and blot' (PAB) approach with magnetic silica particles on filter paper.

    Science.gov (United States)

    Li, Jingyi; Liu, Qian; Alsamarri, Hussein; Lounsbury, Jenny A; Haversitick, Doris M; Landers, James P

    2013-03-07

    Reliable measurement of DNA concentration is essential for a broad range of applications in biology and molecular biology, and for many of these, quantifying the nucleic acid content is inextricably linked to obtaining optimal results. In its most simplistic form, quantitative analysis of nucleic acids can be accomplished by UV-Vis absorbance and, in more sophisticated format, by fluorimetry. A recently reported new concept, the 'pinwheel assay', involves a label-free approach for quantifying DNA through aggregation of paramagnetic beads in a rotating magnetic field. Here, we describe a simplified version of that assay adapted for execution using only a pipet and filter paper. The 'pipette, aggregate, and blot' (PAB) approach allows DNA to induce bead aggregation in a pipette tip through exposure to a magnetic field, followed by dispensing (blotting) onto filter paper. The filter paper immortalises the extent of aggregation, and digital images of the immortalized bead conformation, acquired with either a document scanner or a cell phone camera, allows for DNA quantification using a noncomplex algorithm. Human genomic DNA samples extracted from blood are quantified with the PAB approach and the results utilized to define the volume of sample used in a PCR reaction that is sensitive to input mass of template DNA. Integrating the PAB assay with paper-based DNA extraction and detection modalities has the potential to yield 'DNA quant-on-paper' devices that may be useful for point-of-care testing.

  15. Label-free detection of kanamycin based on a G-quadruplex DNA aptamer-based fluorescent intercalator displacement assay

    Science.gov (United States)

    Xing, Yun-Peng; Liu, Chun; Zhou, Xiao-Hong; Shi, Han-Chang

    2015-01-01

    This work was the first to report that the kanamycin-binding DNA aptamer (5'-TGG GGG TTG AGG CTA AGC CGA-3') can form stable parallel G-quadruplex DNA (G4-DNA) structures by themselves and that this phenomenon can be verified by nondenaturing polyacrylamide gel electrophoresis and circular dichroism spectroscopy. Based on these findings, we developed a novel label-free strategy for kanamycin detection based on the G4-DNA aptamer-based fluorescent intercalator displacement assay with thiazole orange (TO) as the fluorescence probe. In the proposed strategy, TO became strongly fluorescent upon binding to kanamycin-binding G4-DNA. However, the addition of kanamycin caused the displacement of TO from the G4-DNA-TO conjugate, thereby resulting in decreased fluorescent signal, which was inversely related to the kanamycin concentration. The detection limit of the proposed assay decreased to 59 nM with a linear working range of 0.1 μM to 20 μM for kanamycin. The cross-reactivity against six other antibiotics was negligible compared with the response to kanamycin. A satisfactory recovery of kanamycin in milk samples ranged from 80.1% to 98.0%, confirming the potential of this bioassay in the measurement of kanamycin in various applications. Our results also served as a good reference for developing similar fluorescent G4-DNA-based bioassays in the future.

  16. A label-free fluorescent adenosine triphosphate biosensor via overhanging aptamer-triggered enzyme protection and target recycling amplification.

    Science.gov (United States)

    Wang, Zhaoyin; Zhao, Jian; Dai, Zhihui

    2016-06-20

    Herein, a label-free fluorescent adenosine triphosphate (ATP) aptasensor is fabricated with a DNA hairpin and an overhanging aptamer. In the presence of ATP, the overhanging sequences of the aptamer may form preferred substrates of exo III, and thus trigger the enzyme-assisted amplification, which results in the release of G-rich sequences. Free G-rich sequences subsequently generate an enhanced flourescent signal by binding with thioflavin T. However, if ATP is absent, the overhanging sequence can induce steric hindrance and protect the DNA hairpin against the digestion of exo III, significantly reducing the noise of this biosensor. Accordingly, the signal-to-noise ratio of the sensing system is greatly improved, which ensures the desirable analytical performance of the proposed aptasensor both in pure samples and real samples.

  17. Label-free fluorescence strategy for sensitive detection of adenosine triphosphate using a loop DNA probe with low background noise.

    Science.gov (United States)

    Lin, Chunshui; Cai, Zhixiong; Wang, Yiru; Zhu, Zhi; Yang, Chaoyong James; Chen, Xi

    2014-07-15

    A simple, rapid, label-free, and ultrasensitive fluorescence strategy for adenosine triphosphate (ATP) detection was developed using a loop DNA probe with low background noise. In this strategy, a loop DNA probe, which is the substrate for both ligation and digestion enzyme reaction, was designed. SYBR green I (SG I), a double-stranded specific dye, was applied for the readout fluorescence signal. Exonuclease I (Exo I) and exonuclease III (Exo III), sequence-independent nucleases, were selected to digest the loop DNA probe in order to minimize the background fluorescence signal. As a result, in the absence of ATP, the loop DNA was completely digested by Exo I and Exo III, leading to low background fluorescence owing to the weak electrostatic interaction between SG I and mononucleotides. On the other hand, ATP induced the ligation of the nicking site, and the sealed loop DNA resisted the digestion of Exo I and ExoIII, resulting in a remarkable increase of fluorescence response. Upon background noise reduction, the sensitivity of the ATP determination was improved significantly, and the detection limitation was found to be 1.2 pM, which is much lower than that in almost all the previously reported methods. This strategy has promise for wide application in the determination of ATP.

  18. Label-free DNA biosensor based on resistance change of platinum nanoparticles assemblies.

    Science.gov (United States)

    Skotadis, Evangelos; Voutyras, Konstantinos; Chatzipetrou, Marianneza; Tsekenis, Georgios; Patsiouras, Lampros; Madianos, Leonidas; Chatzandroulis, Stavros; Zergioti, Ioanna; Tsoukalas, Dimitris

    2016-07-15

    A novel nanoparticle based biosensor for the fast and simple detection of DNA hybridization events is presented. The sensor utilizes hybridized DNA's charge transport properties, combining them with metallic nanoparticle networks that act as nano-gapped electrodes. The DNA hybridization events can be detected by a significant reduction in the sensor's resistance due to the conductive bridging offered by hybridized DNA. By modifying the nanoparticle surface coverage, which can be controlled experimentally being a function of deposition time, and the structural properties of the electrodes, an optimized biosensor for the in situ detection of DNA hybridization events is ultimately fabricated. The fabricated biosensor exhibits a wide response range, covering four orders of magnitude, a limit of detection of 1nM and can detect a single base pair mismatch between probe and complementary DNA. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. A Microneedle Functionalized with Polyethyleneimine and Nanotubes for Highly Sensitive, Label-Free Quantification of DNA

    OpenAIRE

    Saadat-Moghaddam, Darius; Kim, Jong-Hoon

    2017-01-01

    The accurate measure of DNA concentration is necessary for many DNA-based biological applications. However, the current methods are limited in terms of sensitivity, reproducibility, human error, and contamination. Here, we present a microneedle functionalized with polyethyleneimine (PEI) and single-walled carbon nanotubes (SWCNTs) for the highly sensitive quantification of DNA. The microneedle was fabricated using ultraviolet (UV) lithography and anisotropic etching, and then functionalized w...

  20. A silicon-based electrochemical sensor for highly sensitive, specific, label-free and real-time DNA detection

    International Nuclear Information System (INIS)

    Guo, Yuanyuan; Su, Shao; Wei, Xinpan; Zhong, Yiling; Su, Yuanyuan; He, Yao; Huang, Qing; Fan, Chunhai

    2013-01-01

    We herein present a new kind of silicon-based electrochemical sensor using a gold nanoparticles-decorated silicon wafer (AuNPs@Si) as a high-performance electrode, which is facilely prepared via in situ AuNPs growth on a silicon wafer. Particularly significantly, the resultant electrochemical sensor is efficacious for label-free DNA detection with high sensitivity due to the unique merits of the prepared silicon-based electrode. Typically, DNA at remarkably low concentrations (1–10 fM) could be readily detected without requiring additional signal-amplification procedures, which is better than or comparable to the lowest DNA concentration ever detected via well-studied signal-amplification-assisted electrochemical sensors. Moreover, the silicon-based sensor features high specificity, allowing unambiguous discrimination of single-based mismatches. We further show that real-time DNA assembly is readily monitored via recording the intensity changes of current signals due to the robust thermal stability of the silicon-based electrode. The unprecedented advantages of the silicon-based electrochemical sensor would offer new opportunities for myriad sensing applications. (paper)

  1. Hall effect biosensors with ultraclean graphene film for improved sensitivity of label-free DNA detection

    KAUST Repository

    Loan, Phan Thi Kim; Wu, Dongqin; Ye, Chen; Li, Xiaoqing; Tra, Vu Thanh; Wei, Qiuping; Fu, Li; Yu, Aimin; Li, Lain-Jong; Lin, Cheng-Te

    2017-01-01

    The quality of graphene strongly affects the performance of graphene-based biosensors which are highly demanded for the sensitive and selective detection of biomolecules, such as DNA. This work reported a novel transfer process for preparing a

  2. Homogeneous and label-free electrochemiluminescence aptasensor based on the difference of electrostatic interaction and exonuclease-assisted target recycling amplification.

    Science.gov (United States)

    Ni, Jiancong; Yang, Weiqiang; Wang, Qingxiang; Luo, Fang; Guo, Longhua; Qiu, Bin; Lin, Zhenyu; Yang, Huanghao

    2018-05-15

    The difference of electrostatic interaction between free Ru(phen) 3 2+ and Ru(phen) 3 2+ embedded in double strand DNA (dsDNA) to the negatively charged indium tin oxide (ITO) electrode has been applied to develop a homogeneous and label-free electrochemiluminescence (ECL) aptasensor for the first time. Ochratoxin A (OTA) has been chosen as the model target. The OTA aptamer is first hybridized with its complementary single strand DNA (ssDNA) to form dsDNA and then interacted with Ru(phen) 3 2+ via the grooves binding mode to form dsDNA-Ru(phen) 3 2+ complex, which remains negatively charged feature as well as low diffusion capacity to the negatively charged ITO electrode surface owing to the electrostatic repulsion. Meanwhile, the intercalated Ru(phen) 3 2+ in the grooves of dsDNA works as an ECL signal reporter instead of the labor-intensive labeling steps and can generate much more ECL signal than that from the labeling probe. In the presence of target, the aptamer prefers to form an aptamer-target complex in lieu of dsDNA, which induces the releasing of Ru(phen) 3 2+ from the dsDNA-Ru(phen) 3 2+ complex into the solution. With the assistance of RecJ f exonuclease (a ssDNA specific exonuclease), the released ssDNA and the aptamer in the target-complex were digested into mononucleotides. In the meantime, the target can be also liberated from OTA-aptamer complex and induce target cycling and large amount of free Ru(phen) 3 2+ present in the solution. Since Ru(phen) 3 2+ contains positive charges, which can diffuses easily to the ITO electrode surface because of electrostatic attraction, causing an obviously enhanced ECL signal detected. Under the optimal conditions, the enhanced ECL of the system has a linear relationship with the OTA concentration in the range of 0.01-1.0 ng/mL with a detection limit of 2 pg/mL. This innovative system not only expands the immobilization-free sensors in the electrochemiluminescent fields, but also can be developed for the

  3. A Microneedle Functionalized with Polyethyleneimine and Nanotubes for Highly Sensitive, Label-Free Quantification of DNA.

    Science.gov (United States)

    Saadat-Moghaddam, Darius; Kim, Jong-Hoon

    2017-08-16

    The accurate measure of DNA concentration is necessary for many DNA-based biological applications. However, the current methods are limited in terms of sensitivity, reproducibility, human error, and contamination. Here, we present a microneedle functionalized with polyethyleneimine (PEI) and single-walled carbon nanotubes (SWCNTs) for the highly sensitive quantification of DNA. The microneedle was fabricated using ultraviolet (UV) lithography and anisotropic etching, and then functionalized with PEI and SWCNTs through a dip coating process. The electrical characteristics of the microneedle change with the accumulation of DNA on the surface. Current-voltage measurements in deionized water were conducted to study these changes in the electrical properties of the sensor. The sensitivity test found the signal to be discernable from the noise level down to 100 attomolar (aM), demonstrating higher sensitivity than currently available UV fluorescence and UV absorbance based methods. A microneedle without any surface modification only had a 100 femtomolar (fM) sensitivity. All measurement results were consistent with fluorescence microscopy.

  4. Nanoparticle sensor for label free detection of swine DNA in mixed biological samples

    Energy Technology Data Exchange (ETDEWEB)

    Ali, M E; Hashim, U [Institute of Nano Electronic Engineering (INNE), Universiti Malaysia Perlis, Lot 104-108, Tingkat 1, Block A, Taman Pertiwi Indah, Jalan Kangar-Alor Star, Seriab, 01000 Kangar, Perlis (Malaysia); Mustafa, S; Che Man, Y B; Yusop, M H M [Halal Products Research Institute, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor (Malaysia); Bari, M F [School of Materials Engineering, University Malaysia Perlis, Seriab 01000, Kangar, Perlis (Malaysia); Islam, Kh N [Department of Preclinical Sciences, Faculty of Veterinary Medicine, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor (Malaysia); Hasan, M F, E-mail: uda@unimap.edu.my [Department of Crop Science, Faculty of Agriculture, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor (Malaysia)

    2011-05-13

    We used 40 {+-} 5 nm gold nanoparticles (GNPs) as colorimetric sensor to visually detect swine-specific conserved sequence and nucleotide mismatch in PCR-amplified and non-amplified mitochondrial DNA mixtures to authenticate species. Colloidal GNPs changed color from pinkish-red to gray-purple in 2 mM PBS. Visually observed results were clearly reflected by the dramatic reduction of surface plasmon resonance peak at 530 nm and the appearance of new features in the 620-800 nm regions in their absorption spectra. The particles were stabilized against salt-induced aggregation upon the adsorption of single-stranded DNA. The PCR products, without any additional processing, were hybridized with a 17-base probe prior to exposure to GNPs. At a critical annealing temperature (55 {sup 0}C) that differentiated matched and mismatched base pairing, the probe was hybridized to pig PCR product and dehybridized from the deer product. The dehybridized probe stuck to GNPs to prevent them from salt-induced aggregation and retained their characteristic red color. Hybridization of a 27-nucleotide probe to swine mitochondrial DNA identified them in pork-venison, pork-shad and venison-shad binary admixtures, eliminating the need of PCR amplification. Thus the assay was applied to authenticate species both in PCR-amplified and non-amplified heterogeneous biological samples. The results were determined visually and validated by absorption spectroscopy. The entire assay (hybridization plus visual detection) was performed in less than 10 min. The LOD (for genomic DNA) of the assay was 6 {mu}g ml{sup -1} swine DNA in mixed meat samples. We believe the assay can be applied for species assignment in food analysis, mismatch detection in genetic screening and homology studies between closely related species.

  5. Nanoparticle sensor for label free detection of swine DNA in mixed biological samples

    International Nuclear Information System (INIS)

    Ali, M E; Hashim, U; Mustafa, S; Che Man, Y B; Yusop, M H M; Bari, M F; Islam, Kh N; Hasan, M F

    2011-01-01

    We used 40 ± 5 nm gold nanoparticles (GNPs) as colorimetric sensor to visually detect swine-specific conserved sequence and nucleotide mismatch in PCR-amplified and non-amplified mitochondrial DNA mixtures to authenticate species. Colloidal GNPs changed color from pinkish-red to gray-purple in 2 mM PBS. Visually observed results were clearly reflected by the dramatic reduction of surface plasmon resonance peak at 530 nm and the appearance of new features in the 620-800 nm regions in their absorption spectra. The particles were stabilized against salt-induced aggregation upon the adsorption of single-stranded DNA. The PCR products, without any additional processing, were hybridized with a 17-base probe prior to exposure to GNPs. At a critical annealing temperature (55 0 C) that differentiated matched and mismatched base pairing, the probe was hybridized to pig PCR product and dehybridized from the deer product. The dehybridized probe stuck to GNPs to prevent them from salt-induced aggregation and retained their characteristic red color. Hybridization of a 27-nucleotide probe to swine mitochondrial DNA identified them in pork-venison, pork-shad and venison-shad binary admixtures, eliminating the need of PCR amplification. Thus the assay was applied to authenticate species both in PCR-amplified and non-amplified heterogeneous biological samples. The results were determined visually and validated by absorption spectroscopy. The entire assay (hybridization plus visual detection) was performed in less than 10 min. The LOD (for genomic DNA) of the assay was 6 μg ml -1 swine DNA in mixed meat samples. We believe the assay can be applied for species assignment in food analysis, mismatch detection in genetic screening and homology studies between closely related species.

  6. Target recycling amplification for label-free and sensitive colorimetric detection of adenosine triphosphate based on un-modified aptamers and DNAzymes.

    Science.gov (United States)

    Gong, Xue; Li, Jinfu; Zhou, Wenjiao; Xiang, Yun; Yuan, Ruo; Chai, Yaqin

    2014-05-30

    Based on target recycling amplification, the development of a new label-free, simple and sensitive colorimetric detection method for ATP by using un-modified aptamers and DNAzymes is described. The association of the model target molecules (ATP) with the corresponding aptamers of the dsDNA probes leads to the release of the G-quadruplex sequences. The ATP-bound aptamers can be further degraded by Exonuclease III to release ATP, which can again bind the aptamers of the dsDNA probes to initiate the target recycling amplification process. Due to this target recycling amplification, the amount of the released G-quadruplex sequences is significantly enhanced. Subsequently, these G-quadruplex sequences bind hemin to form numerous peroxidase mimicking DNAzymes, which cause substantially intensified color change of the probe solution for highly sensitive colorimetric detection of ATP down to the sub-nanomolar (0.33nM) level. Our method is highly selective toward ATP against other control molecules and can be performed in one single homogeneous solution, which makes our sensing approach hold great potential for sensitive colorimetric detection of other small molecules and proteins. Copyright © 2014 Elsevier B.V. All rights reserved.

  7. Label-free DNA electrochemical sensor based on a PNA-functionalized conductive polymer

    DEFF Research Database (Denmark)

    Reisberg, S; Dang, L A; Nguyen, Q A

    2008-01-01

    -solution interface. A reagentless and direct electrochemical detection was obtained by detection of the electrochemical changes using square wave voltammetry (SWV). An increase in the peak current of quinone was observed upon hybridization of probe on the target, whereas no change is observed with non...

  8. Label-free and ultrasensitive electrochemiluminescence detection of microRNA based on long-range self-assembled DNA nanostructures

    International Nuclear Information System (INIS)

    Liu, Ting; Chen, Xian; Hong, Cheng-Yi; Xu, Xiao-Ping; Yang, Huang-Hao

    2014-01-01

    Electrochemiluminescence (ECL) integrates the advantages of electrochemical detection and chemiluminescent techniques. The method has received particular attention because it is highly sensitive and selective, has a wide linear range but low reagent costs. The use of nanomaterials with their unique physical and chemical properties has led to new kinds of biosensors that exhibit high sensitivity and stability. Compared to other nanomaterials, DNA nanostructures are more biocompatible, more hydrophilic, and thus less prone to nonspecific adsorption onto the electrode surface. We describe here a label-free and ultrasensitive ECL biosensor for detecting a cancer-associated microRNA at a femtomolar level. We have designed two auxiliary probes that cause the formation of a long-range self-assembly in the form of a μm-long 1-dimensional DNA concatamer. These can be used as carriers for signal amplification. The intercalation of the ECL probe Ru(phen) 3 2+ into the grooves of the concatamers leads to a substantial increase in ECL intensity. This amplified sensor shows high selectivity for discriminating complementary target and other mismatched RNAs. The biosensor enables the quantification of the expression of microRNA-21 in MCF-7 cells. It also displays very low limits of detection and provides an alternative approach for the detection of RNA or DNA detection in diagnostics and gene analysis. (author)

  9. Label-free, electrochemical detection of methicillin-resistant staphylococcus aureus DNA with reduced graphene oxide-modified electrodes

    KAUST Repository

    Wang, Zhijuan

    2011-05-01

    Reduced graphene oxide (rGO)-modified glassy carbon electrode is used to detect the methicillin-resistant Staphylococcus aureus (MRSA) DNA by using electrochemical impedance spectroscopy. Our experiments confirm that ssDNA, before and after hybridization with target DNA, are successfully anchored on the rGO surface. After the probe DNA, pre-adsorbed on rGO electrode, hybridizes with target DNA, the measured impedance increases dramatically. It provides a new method to detect DNA with high sensitivity (10-13M, i.e., 100 fM) and selectivity. © 2011 Elsevier B.V.

  10. One-pot synthesis of strongly fluorescent DNA-CuInS2 quantum dots for label-free and ultrasensitive detection of anthrax lethal factor DNA

    International Nuclear Information System (INIS)

    Liu, Ziping; Su, Xingguang

    2016-01-01

    Herein, high quality DNA-CuInS 2 QDs are facilely synthesized through a one-pot hydrothermal method with fluorescence quantum yield as high as 23.4%, and the strongly fluorescent DNA-CuInS 2 QDs have been utilized as a novel fluorescent biosensor for label-free and ultrasensitive detection of anthrax lethal factor DNA. L-Cysteine (L-Cys) and a specific-sequence DNA are used as co-ligands to stabilize the CuInS 2 QDs. The specific-sequence DNA consists of two domains: phosphorothiolates domain (sulfur-containing variants of the usual phosphodiester backbone) controls the nanocrystal passivation and serves as a ligand, and the functional domain (non-phosphorothioates) controls the biorecognition. The as-prepared DNA-CuInS 2 QDs have high stability, good water-solubility and low toxicity. Under the optimized conditions, a linear correlation was established between the fluorescence intensity ratio I/I 0 (I 0 is the original fluorescence intensity of DNA-CuInS 2 QDs, and I is the fluorescence intensity of DNA-CuInS 2 QDs/GO with the addition of various concentrations of anthrax lethal factor DNA) and the concentration of anthrax lethal factor DNA in the range of 0.029–0.733 nmol L −1 with a detection limit of 0.013 nmol L −1 . The proposed method has been successfully applied to the determination of anthrax lethal factor DNA sequence in human serum samples with satisfactory results. Because of low toxicity and fine biocompatibility, DNA-CuInS 2 QDs also hold potential applications in bioimaging. - Highlights: • Strongly fluorescent DNA-QDs were successfully prepared by a one-pot hydrothermal method with quantum yield up to 23.4%. • A biosensor for label-free detection of anthrax lethal factor DNA was established based on the as-prepared DNA-QDs. • The DNA sensor took advantage of the feature that ssDNA binds to GO with significantly higher affinity than dsDNA. • Good sensitivity and selectivity were obtained. • This method was utilized to detect

  11. One-pot synthesis of strongly fluorescent DNA-CuInS{sub 2} quantum dots for label-free and ultrasensitive detection of anthrax lethal factor DNA

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Ziping; Su, Xingguang, E-mail: suxg@jlu.edu.cn

    2016-10-26

    Herein, high quality DNA-CuInS{sub 2} QDs are facilely synthesized through a one-pot hydrothermal method with fluorescence quantum yield as high as 23.4%, and the strongly fluorescent DNA-CuInS{sub 2} QDs have been utilized as a novel fluorescent biosensor for label-free and ultrasensitive detection of anthrax lethal factor DNA. L-Cysteine (L-Cys) and a specific-sequence DNA are used as co-ligands to stabilize the CuInS{sub 2} QDs. The specific-sequence DNA consists of two domains: phosphorothiolates domain (sulfur-containing variants of the usual phosphodiester backbone) controls the nanocrystal passivation and serves as a ligand, and the functional domain (non-phosphorothioates) controls the biorecognition. The as-prepared DNA-CuInS{sub 2} QDs have high stability, good water-solubility and low toxicity. Under the optimized conditions, a linear correlation was established between the fluorescence intensity ratio I/I{sub 0} (I{sub 0} is the original fluorescence intensity of DNA-CuInS{sub 2} QDs, and I is the fluorescence intensity of DNA-CuInS{sub 2} QDs/GO with the addition of various concentrations of anthrax lethal factor DNA) and the concentration of anthrax lethal factor DNA in the range of 0.029–0.733 nmol L{sup −1} with a detection limit of 0.013 nmol L{sup −1}. The proposed method has been successfully applied to the determination of anthrax lethal factor DNA sequence in human serum samples with satisfactory results. Because of low toxicity and fine biocompatibility, DNA-CuInS{sub 2} QDs also hold potential applications in bioimaging. - Highlights: • Strongly fluorescent DNA-QDs were successfully prepared by a one-pot hydrothermal method with quantum yield up to 23.4%. • A biosensor for label-free detection of anthrax lethal factor DNA was established based on the as-prepared DNA-QDs. • The DNA sensor took advantage of the feature that ssDNA binds to GO with significantly higher affinity than dsDNA. • Good sensitivity and selectivity were

  12. Disulfide-induced self-assembled targets : A novel strategy for the label free colorimetric detection of DNAs/RNAs via unmodified gold nanoparticles

    NARCIS (Netherlands)

    Shokri, Ehsan; Hosseini, Morteza; Davari, Mehdi D.; Ganjali, Mohammad R.; Peppelenbosch, Maikel P.; Rezaee, Farhad

    2017-01-01

    A modified non-cross-linking gold-nanoparticles (Au-NPs) aggregation strategy has been developed for the label free colorimetric detection of DNAs/RNAs based on self-assembling target species in the presence of thiolated probes. Two complementary thiol-modified probes, each of which specifically

  13. Disulfide-induced self-assembled targets: A novel strategy for the label free colorimetric detection of DNAs/RNAs via unmodified gold nanoparticles

    NARCIS (Netherlands)

    Shokri, E. (Ehsan); M. Hosseini (Morteza); Davari, M.D. (Mehdi D.); Ganjali, M.R. (Mohammad R.); M.P. Peppelenbosch (Maikel); F. Rezaee (Farhad)

    2017-01-01

    textabstractA modified non-cross-linking gold-nanoparticles (Au-NPs) aggregation strategy has been developed for the label free colorimetric detection of DNAs/RNAs based on self-assembling target species in the presence of thiolated probes. Two complementary thiol- modified probes, each of which

  14. Cascaded strand displacement for non-enzymatic target recycling amplification and label-free electronic detection of microRNA from tumor cells

    Energy Technology Data Exchange (ETDEWEB)

    Shi, Kai; Dou, Baoting; Yang, Jianmei; Yuan, Ruo; Xiang, Yun, E-mail: yunatswu@swu.edu.cn

    2016-04-15

    The monitoring of microRNA (miRNA) expression levels is of great importance in cancer diagnosis. In the present work, based on two cascaded toehold-mediated strand displacement reactions (TSDRs), we have developed a label- and enzyme-free target recycling signal amplification approach for sensitive electronic detection of miRNA-21 from human breast cancer cells. The junction probes containing the locked G-quadruplex forming sequences are self-assembled on the senor surface. The presence of the target miRNA-21 initiates the first TSDR and results in the disassembly of the junction probes and the release of the active G-quadruplex forming sequences. Subsequently, the DNA fuel strand triggers the second TSDR and leads to cyclic reuse of the target miRNA-21. The cascaded TSDRs thus generate many active G-quadruplex forming sequences on the sensor surface, which associate with hemin to produce significantly amplified current response for sensitive detection of miRNA-21 at 1.15 fM. The sensor is also selective and can be employed to monitor miRNA-21 from human breast cancer cells. - Highlights: • Amplified and sensitive detection of microRNA from tumor cells is achieved. • Signal amplification is realized by two cascaded strand displacement reactions. • The developed sensor is selective and label-free without involving any enzymes.

  15. Label-Free and Ultrasensitive Biomolecule Detection Based on Aggregation Induced Emission Fluorogen via Target-Triggered Hemin/G-Quadruplex-Catalyzed Oxidation Reaction.

    Science.gov (United States)

    Li, Haiyin; Chang, Jiafu; Gai, Panpan; Li, Feng

    2018-02-07

    Fluorescence biosensing strategy has drawn substantial attention due to their advantages of simplicity, convenience, sensitivity, and selectivity, but unsatisfactory structure stability, low fluorescence quantum yield, high cost of labeling, and strict reaction conditions associated with current fluorescence methods severely prohibit their potential application. To address these challenges, we herein propose an ultrasensitive label-free fluorescence biosensor by integrating hemin/G-quadruplex-catalyzed oxidation reaction with aggregation induced emission (AIE) fluorogen-based system. l-Cysteine/TPE-M, which is carefully and elaborately designed and developed, obviously contributes to strong fluorescence emission. In the presence of G-rich DNA along with K + and hemin, efficient destruction of l-cysteine occurs due to hemin/G-quadruplex-catalyzed oxidation reactions. As a result, highly sensitive fluorescence detection of G-rich DNA is readily realized, with a detection limit down to 33 pM. As a validation for the further development of the proposed strategy, we also successfully construct ultrasensitive platforms for microRNA by incorporating the l-cysteine/TPE-M system with target-triggered cyclic amplification reaction. Thus, this proposed strategy is anticipated to find use in basic biochemical research and clinical diagnosis.

  16. Disulfide-induced self-assembled targets: A novel strategy for the label free colorimetric detection of DNAs/RNAs via unmodified gold nanoparticles

    Science.gov (United States)

    Shokri, Ehsan; Hosseini, Morteza; Davari, Mehdi D.; Ganjali, Mohammad R.; Peppelenbosch, Maikel P.; Rezaee, Farhad

    2017-04-01

    A modified non-cross-linking gold-nanoparticles (Au-NPs) aggregation strategy has been developed for the label free colorimetric detection of DNAs/RNAs based on self-assembling target species in the presence of thiolated probes. Two complementary thiol- modified probes, each of which specifically binds at one half of the target introduced SH groups at both ends of dsDNA. Continuous disulfide bond formation at 3‧ and 5‧ terminals of targets leads to the self-assembly of dsDNAs into the sulfur- rich and flexible products with different lengths. These products have a high affinity for the surface of Au-NPs and efficiently protect the surface from salt induced aggregation. To evaluate the assay efficacy, a small part of the citrus tristeza virus (CTV) genome was targeted, leading to a detection limit of about 5 × 10-9 mol.L-1 over a linear ranged from 20 × 10-9 to 10 × 10-7 mol.L-1. This approach also exhibits good reproducibility and recovery levels in the presence of plant total RNA or human plasma total circulating RNA extracts. Self-assembled targets can be then sensitively distinguished from non-assembled or mismatched targets after gel electrophoresis. The disulfide reaction method and integrating self-assembled DNAs/RNAs targets with bare AuNPs as a sensitive indicator provide us a powerful and simple visual detection tool for a wide range of applications.

  17. The beauty of being (label)-free: sample preparation methods for SWATH-MS and next-generation targeted proteomics

    Science.gov (United States)

    Campbell, Kate; Deery, Michael J.; Lilley, Kathryn S.; Ralser, Markus

    2014-01-01

    The combination of qualitative analysis with label-free quantification has greatly facilitated the throughput and flexibility of novel proteomic techniques. However, such methods rely heavily on robust and reproducible sample preparation procedures. Here, we benchmark a selection of in gel, on filter, and in solution digestion workflows for their application in label-free proteomics. Each procedure was associated with differing advantages and disadvantages. The in gel methods interrogated were cost effective, but were limited in throughput and digest efficiency. Filter-aided sample preparations facilitated reasonable processing times and yielded a balanced representation of membrane proteins, but led to a high signal variation in quantification experiments. Two in solution digest protocols, however, gave optimal performance for label-free proteomics. A protocol based on the detergent RapiGest led to the highest number of detected proteins at second-best signal stability, while a protocol based on acetonitrile-digestion, RapidACN, scored best in throughput and signal stability but came second in protein identification. In addition, we compared label-free data dependent (DDA) and data independent (SWATH) acquisition on a TripleTOF 5600 instrument. While largely similar in protein detection, SWATH outperformed DDA in quantification, reducing signal variation and markedly increasing the number of precisely quantified peptides. PMID:24741437

  18. Label-free colorimetric detection of Hg²⁺ based on Hg²⁺-triggered exonuclease III-assisted target recycling and DNAzyme amplification.

    Science.gov (United States)

    Ren, Wang; Zhang, Ying; Huang, Wei Tao; Li, Nian Bing; Luo, Hong Qun

    2015-06-15

    This work reported a label-free colorimetric assay for sensitive detection of Hg(2+) based on Hg(2+)-triggered hairpin DNA probe (H-DNA) termini-binding and exonuclease Ш (Exo Ш)-assisted target recycling, as well as hemin/G-quadruplex (DNAzyme) signal amplification. The specific binding of free Hg(2+) with the thymine-thymine (T-T) mismatches termini of H-DNA could immediately trigger the Exo Ш digestion, and then set free G-quadruplex segments and Hg(2+). The Exo Ш impellent recycling of ultratrace Hg(2+) produced numerous G-quadruplexes. The corresponding DNAzymes catalyzed efficiently the H2O2-mediated oxidation of the ABTS(2-) to the colored product in the presence of hemin. Using the color change as the output signal, and the Exo Ш-aided Hg(2+) recycling and DNAzyme as the signal amplifier, the ultrasensitive assay system successfully achieved visual detection of Hg(2+) as low as 1.0 nM by the naked eye, and was suitable for field monitoring. The calibration curve was linear in the range of 50.0 pM to 20.0 nM for Hg(2+) (R=0.9962) with a detection limit of 10.0 pM. Moreover, this proposed strategy showed excellent selectivity, portability and low-cost, and was successfully applied to colorimetric detection of Hg(2+) in laboratory tap water and Jialing river water samples. Copyright © 2015 Elsevier B.V. All rights reserved.

  19. Label-free DNA hybridization detection and single base-mismatch discrimination using CE-ICP-MS assay.

    Science.gov (United States)

    Li, Yan; Sun, Shao-kai; Yang, Jia-lin; Jiang, Yan

    2011-12-07

    Detecting a specific DNA sequence and discriminating single base-mismatch is critical to clinical diagnosis, paternity testing, forensic sciences, food and drug industry, pathology, genetics, environmental monitoring, and anti-bioterrorism. To this end, capillary electrophoresis (CE) coupled with the inductively coupled plasma mass spectrometry (ICP-MS) method is developed using the displacing interaction between the target ssDNA and the competitor Hg(2+) for the first time. The thymine-rich capture ssDNA 1 is interacted with the competitor Hg(2+), forming an assembled complex in a hairpin-structure between the thymine bases arrangement at both sides of the capture ssDNA 1. In the presence of a target ssDNA with stronger affinity than that of the competitor Hg(2+), the energetically favorable hybridization between capture ssDNA 1 and the target ssDNA destroys the hairpin-structure and releases the competitor as free Hg(2+), which was then read out and accurately quantified by CE-ICP-MS assay. Under the optimal CE separation conditions, free Hg(2+) ions and its capture ssDNA 1 adduct were baseline separated and detected on-line by ICP-MS; the increased peak intensity of free Hg(2+) against the concentration of perfectly complementary target ssDNA was linear over the concentration range of 30-600 nmol L(-1) with a limit of detection of 8 nmol L(-1) (3s, n = 11) in the pre-incubated mixture containing 1 μmol L(-1) Hg(2+) and 0.2 μmol L(-1) capture ssDNA 1. This new assay method is simple in design since any target ssDNA binding can in principle result in free Hg(2+) release by 6-fold Hg(2+) signal amplification, avoiding oligonucleotide labeling or assistance by excess signal transducer and signal reporter to read out the target. Due to element-specific detection of ICP-MS in our assay procedure, the interference from the autofluorescence of substrata was eliminated.

  20. Low-cost label-free electrical detection of artificial DNA nanostructures using solution-processed oxide thin-film transistors.

    Science.gov (United States)

    Kim, Si Joon; Jung, Joohye; Lee, Keun Woo; Yoon, Doo Hyun; Jung, Tae Soo; Dugasani, Sreekantha Reddy; Park, Sung Ha; Kim, Hyun Jae

    2013-11-13

    A high-sensitivity, label-free method for detecting deoxyribonucleic acid (DNA) using solution-processed oxide thin-film transistors (TFTs) was developed. Double-crossover (DX) DNA nanostructures with different concentrations of divalent Cu ion (Cu(2+)) were immobilized on an In-Ga-Zn-O (IGZO) back-channel surface, which changed the electrical performance of the IGZO TFTs. The detection mechanism of the IGZO TFT-based DNA biosensor is attributed to electron trapping and electrostatic interactions caused by negatively charged phosphate groups on the DNA backbone. Furthermore, Cu(2+) in DX DNA nanostructures generates a current path when a gate bias is applied. The direct effect on the electrical response implies that solution-processed IGZO TFTs could be used to realize low-cost and high-sensitivity DNA biosensors.

  1. Label-free and sensitive detection of T4 polynucleotide kinase activity via coupling DNA strand displacement reaction with enzymatic-aided amplification.

    Science.gov (United States)

    Cheng, Rui; Tao, Mangjuan; Shi, Zhilu; Zhang, Xiafei; Jin, Yan; Li, Baoxin

    2015-11-15

    Several fluorescence signal amplification strategies have been developed for sensitive detection of T4 polynucleotide kinase (T4 PNK) activity, but they need fluorescence dye labeled DNA probe. We have addressed the limitation and report here a label-free strategy for sensitive detection of PNK activity by coupling DNA strand displacement reaction with enzymatic-aided amplification. A hairpin oligonucleotide (hpDNA) with blunt ends was used as the substrate for T4 PNK phosphorylation. In the presence of T4 PNK, the stem of hpDNA was phosphorylated and further degraded by lambda exonuclease (λ exo) from 5' to 3' direction to release a single-stranded DNA as a trigger of DNA strand displacement reaction (SDR). The trigger DNA can continuously displace DNA P2 from P1/P2 hybrid with the help of specific cleavage of nicking endonuclease (Nt.BbvCI). Then, DNA P2 can form G-quadruplex in the presence of potassium ions and quadruplex-selective fluorphore, N-methyl mesoporphyrin IX (NMM), resulting in a significant increase in fluorescence intensity of NMM. Thus, the accumulative release of DNA P2 led to fluorescence signal amplification for determining T4 PNK activity with a detection limit of 6.6×10(-4) U/mL, which is superior or comparative with established approaches. By ingeniously utilizing T4 PNK-triggered DNA SDR, T4 PNK activity can be specifically and facilely studied in homogeneous solution containing complex matrix without any external fluorescence labeling. Moreover, the influence of different inhibitors on the T4 PNK activity revealed that it also can be explored to screen T4 PNK inhibitors. Therefore, this label-free amplification strategy presents a facile and cost-effective approach for nucleic acid phosphorylation related research. Copyright © 2015 Elsevier B.V. All rights reserved.

  2. Targeted proteomics guided by label-free global proteome analysis in saliva reveal transition signatures from health to periodontal disease.

    Science.gov (United States)

    Bostanci, Nagihan; Selevsek, Nathalie; Wolski, Witold; Grossmann, Jonas; Bao, Kai; Wahlander, Asa; Trachsel, Christian; Schlapbach, Ralph; Özturk, Veli Özgen; Afacan, Beral; Emingil, Gulnur; Belibasakis, Georgios N

    2018-04-02

    Periodontal diseases are among the most prevalent worldwide, but largely silent, chronic diseases. They affect the tooth-supporting tissues with multiple ramifications on life quality. Their early diagnosis is still challenging, due to lack of appropriate molecular diagnostic methods. Saliva offers a non-invasively collectable reservoir of clinically relevant biomarkers, which, if utilized efficiently, could facilitate early diagnosis and monitoring of ongoing disease. Despite several novel protein markers being recently enlisted by discovery proteomics, their routine diagnostic application is hampered by the lack of validation platforms that allow for rapid, accurate and simultaneous quantification of multiple proteins in large cohorts. We carried out a pipeline of two proteomic platforms; firstly, we applied open ended label-free quantitative (LFQ) proteomics for discovery in saliva (n=67, health, gingivitis, and periodontitis), followed by selected-reaction monitoring (SRM)-targeted proteomics for validation in an independent cohort (n=82). The LFQ platform led to the discovery of 119 proteins with at least two-fold significant difference between health and disease. The 65 proteins chosen for the subsequent SRM platform included 50 related proteins derived from the significantly enriched processes of the LFQ data, 11 from literature-mining, and four house-keeping ones. Among those, 60 were reproducibly quantifiable proteins (92% success rate), represented by a total of 143 peptides. Machine-learning modeling led to a narrowed-down panel of five proteins of high predictive value for periodontal diseases (higher in disease: Matrix metalloproteinase-9, Ras-related protein-1, Actin-related protein 2/3 complex subunit 5; lower in disease: Clusterin, Deleted in Malignant Brain Tumors 1), with maximum area under the receiver operating curve >0.97. This panel enriches the pool of credible clinical biomarker candidates for diagnostic assay development. Yet, the quantum

  3. A quantum dot-aptamer beacon using a DNA intercalating dye as the FRET reporter: application to label-free thrombin detection.

    Science.gov (United States)

    Chi, Chun-Wei; Lao, Yeh-Hsing; Li, Yi-Shan; Chen, Lin-Chi

    2011-03-15

    A new quantum dot (QD)-aptamer (apt) beacon that acts by folding-induced dissociation of a DNA intercalating dye, BOBO-3(B), is demonstrated with label-free thrombin detection. The beacon, denoted as QD-apt:B, is constructed by (1) coupling of a single-stranded thrombin aptamer to Qdot 565 via EDC/Sulfo-NHS chemistry and (2) staining the duplex regions of the aptamer on QD with excess BOBO-3 before thrombin binding. When mixing a thrombin sample with QD-apt:B, BOBO-3 is competed away from the beacon due to target-induced aptamer folding, which then causes a decrease in QD fluorescence resonance energy transfer (FRET)-mediated BOBO-3 emission and achieves thrombin quantitation. In this work, the effects of Mg(2+), coupling time, and aptamer type on the beacon's performances are investigated and discussed thoroughly with various methods, including transmission electron microscopy (TEM), dynamic light scattering (DLS), and two-color differential gel electrophoresis. Using the best aptamer beacon (HTQ37), we attain highly specific and wide-range detection (from nM to μM) of thrombin in buffer, and the beacon can sense nM-range thrombin in 15% diluted serum. Compared to the reported QD aptamer assays, our method is advantageous from the aspect of using a simple sensory unit design without losing the detection sensitivity. Therefore, we consider the QD-apt:B beacon a potential alternative to immuno-reagents and an effective tool to study nucleic acid folding on QD as well. Copyright © 2011 Elsevier B.V. All rights reserved.

  4. A highly sensitive label-free electrochemical aptasensor for interferon-gamma detection based on graphene controlled assembly and nuclease cleavage-assisted target recycling amplification.

    Science.gov (United States)

    Yan, Genping; Wang, Yonghong; He, Xiaoxiao; Wang, Kemin; Liu, Jinquan; Du, Yudan

    2013-06-15

    We report here a highly sensitive and label-free electrochemical aptasensing technology for detection of interferon-gamma (IFN-γ) based on graphene controlled assembly and enzyme cleavage-assisted target recycling amplification strategy. In this work, in the absence of IFN-γ, the graphene could not be assembled onto the 16-mercaptohexadecanoic acid (MHA) modified gold electrode because the IFN-γ binding aptamer was strongly adsorbed on the graphene due to the strong π-π interaction. Thus the electronic transmission was blocked (eT OFF). However, the presence of target IFN-γ and DNase I led to desorption of aptamer from the graphene surface and further cleavage of the aptamer, thereby releasing the IFN-γ. The released IFN-γ could then re-attack other aptamers on the graphene, resulting in the successive release of the aptamers from the graphene. At the same time, the "naked" graphene could be assembled onto the MHA modified gold electrode with hydrophobic interaction and π-conjunction, mediating the electron transfer between the electrode and the electroactive indicator. Then, measurable electrochemical signals were generated (eT ON), which was related to the concentration of the IFN-γ. By taking advantages of graphene and enzyme cleavage-assisted target recycling amplification, the developed label-free electrochemical aptasensing technology showed a linear response to concentration of IFN-γ range from 0.1 to 0.7 pM. The detection limit of IFN-γ was determined to be 0.065 pM. Moreover, this aptasensor shows good selectivity toward the target in the presence of other relevant proteins. Our strategy thus opens new opportunities for label-free and amplified detection of other kinds of proteins. Copyright © 2013 Elsevier B.V. All rights reserved.

  5. Target-induced structure switching of hairpin aptamers for label-free and sensitive fluorescent detection of ATP via exonuclease-catalyzed target recycling amplification.

    Science.gov (United States)

    Xu, Yunying; Xu, Jin; Xiang, Yun; Yuan, Ruo; Chai, Yaqin

    2014-01-15

    In this work, we described the development of a new label-free, simple and sensitive fluorescent ATP sensing platform based on exonuclease III (Exo III)-catalyzed target recycling (ECTR) amplification and SYBR Green I indicator. The hairpin aptamer probes underwent conformational structure switching and re-configuration in the presence of ATP, which led to catalytic cleavage of the re-configured aptamers by Exo III to release ATP and to initiate the ECTR process. Such ECTR process resulted in the digestion of a significant number of the hairpin aptamer probes, leading to much less intercalation of SYBR Green I to the hairpin stems and drastic suppression of the fluorescence emission for sensitive ATP detection down to the low nanomolar level. Due to the highly specific affinity bindings between aptamers and ATP, the developed method exhibited excellent selectivity toward ATP against other analogous molecules. Besides, our ATP sensing approach used un-modified aptamer probes and could be performed in a "mix-and-detect" fashion in homogenous solutions. All these distinct advantages of the developed method thus made it hold great potential for the development of simple and robust sensing strategies for the detection of other small molecules. © 2013 Elsevier B.V. All rights reserved.

  6. Smart Sensing Based on DNA-Metal Interaction Enables a Label-Free and Resettable Security Model of Electrochemical Molecular Keypad Lock.

    Science.gov (United States)

    Du, Yan; Han, Xu; Wang, Chenxu; Li, Yunhui; Li, Bingling; Duan, Hongwei

    2018-01-26

    Recently, molecular keypad locks have received increasing attention. As a new subgroup of smart biosensors, they show great potential for protecting information as a molecular security data processor, rather than merely molecular recognition and quantitation. Herein, label-free electrochemically transduced Ag + and cysteine (Cys) sensors were developed. A molecular keypad lock model with reset function was successfully realized based on the balanced interaction of metal ion with its nucleic acid and chemical ligands. The correct input of "1-2-3" (i.e., "Ag + -Cys-cDNA") is the only password of such molecular keypad lock. Moreover, the resetting process of either correct or wrong input order could be easily made by Cys, buffer, and DI water treatment. Therefore, our system provides an even smarter system of molecular keypad lock, which could inhibit illegal access of unauthorized users, holding great promise in information protection at the molecular level.

  7. Signal-on electrochemical assay for label-free detection of TdT and BamHI activity based on grown DNA nanowire-templated copper nanoclusters.

    Science.gov (United States)

    Hu, Yufang; Zhang, Qingqing; Xu, Lihua; Wang, Jiao; Rao, Jiajia; Guo, Zhiyong; Wang, Sui

    2017-11-01

    Electrochemical methods allow fast and inexpensive analysis of enzymatic activity. Here, a simple and yet efficient "signal-on" electrochemical assay for sensitive, label-free detection of DNA-related enzyme activity was established on the basis of terminal deoxynucleotidyl transferase (TdT)-mediated extension strategy. TdT, which is a template-independent DNA polymerase, can catalyze the sequential addition of deoxythymidine triphosphate (dTTP) at the 3'-OH terminus of single-stranded DNA (ssDNA); then, the TdT-yield T-rich DNA nanowires can be employed as the synthetic template of copper nanoclusters (CuNCs). Grown DNA nanowires-templated CuNCs (noted as DNA-CuNCs) were attached onto graphene oxide (GO) surface and exhibited unique electrocatalytic activity to H 2 O 2 reduction. Under optimal conditions, the proposed biosensor was utilized for quantitatively monitoring TdT activity, with the observed LOD of 0.1 U/mL. It also displayed high selectivity to TdT with excellent stability, and offered a facile, convenient electrochemical method for TdT-relevant inhibitors screening. Moreover, the proposed sensor was successfully used for BamHI activity detection, in which a new 3'-OH terminal was exposed by the digestion of a phosphate group. Ultimately, it has good prospects in DNA-related enzyme-based biochemical studies, disease diagnosis, and drug discovery. Graphical Abstract Extraordinary TdT-generated DNA-CuNCs are synthesized and act as a novel electrochemical sensing platform for sensitive detection of TdT and BamHI activity in biological environments.

  8. Gold nano particle decorated graphene core first generation PAMAM dendrimer for label free electrochemical DNA hybridization sensing.

    Science.gov (United States)

    Jayakumar, K; Rajesh, R; Dharuman, V; Venkatasan, R; Hahn, J H; Pandian, S Karutha

    2012-01-15

    A novel first generation (G1) poly(amidoamine) dendrimer (PAMAM) with graphene core (GG1PAMAM) was synthesized for the first time. Single layer of GG1PAMAM was immobilized covalently on mercaptopropionic acid (MPA) monolayer on Au transducer. This allows cost effective and easy deposition of single layer graphene on the Au transducer surface than the advanced vacuum techniques used in the literature. Au nano particles (17.5 nm) then decorated the GG1PAMAM and used for electrochemical DNA hybridization sensing. The sensor discriminates selectively and sensitively the complementary double stranded DNA (dsDNA, hybridized), non-complementary DNA (ssDNA, un-hybridized) and single nucleotide polymorphism (SNP) surfaces. Interactions of the MPA, GG1PAMAM and the Au nano particles were characterized by Ultra Violet (UV), Fourier Transform Infrared (FTIR), Raman spectroscopy (RS), Thermo gravimetric analysis (TGA), Scanning Electron Microscopy (SEM), Atomic Force Microscopy (AFM), Cyclic Voltmetric (CV), Impedance spectroscopy (IS) and Differntial Pulse Voltammetry (DPV) techniques. The sensor showed linear range 1×10(-6) to 1×10(-12) M with lowest detection limit 1 pM which is 1000 times lower than G1PAMAM without graphene core. Copyright © 2011 Elsevier B.V. All rights reserved.

  9. Ultrasensitive label-free detection of DNA hybridization by sapphire-based graphene field-effect transistor biosensor

    Science.gov (United States)

    Xu, Shicai; Jiang, Shouzhen; Zhang, Chao; Yue, Weiwei; Zou, Yan; Wang, Guiying; Liu, Huilan; Zhang, Xiumei; Li, Mingzhen; Zhu, Zhanshou; Wang, Jihua

    2018-01-01

    Graphene has attracted much attention in biosensing applications for its unique properties. Because of one-atom layer structure, every atom of graphene is exposed to the environment, making the electronic properties of graphene are very sensitive to charged analytes. Therefore, graphene is an ideal material for transistors in high-performance sensors. Chemical vapor deposition (CVD) method has been demonstrated the most successful method for fabricating large area graphene. However, the conventional CVD methods can only grow graphene on metallic substrate and the graphene has to be transferred to the insulating substrate for further device fabrication. The transfer process creates wrinkles, cracks, or tears on the graphene, which severely degrade electrical properties of graphene. These factors severely degrade the sensing performance of graphene. Here, we directly fabricated graphene on sapphire substrate by high temperature CVD without the use of metal catalysts. The sapphire-based graphene was patterned and make into a DNA biosensor in the configuration of field-effect transistor. The sensors show high performance and achieve the DNA detection sensitivity as low as 100 fM (10-13 M), which is at least 10 times lower than prior transferred CVD G-FET DNA sensors. The use of the sapphire-based G-FETs suggests a promising future for biosensing applications.

  10. Label-free and reagentless electrochemical detection of PCR fragments using self-assembled quinone derivative monolayer: Application to Mycobacterium tuberculosis

    DEFF Research Database (Denmark)

    Zhang, Q D; March, G; Noel, V

    2012-01-01

    We report a signal-on, label-free and reagentless electrochemical DNA biosensor, based on a mixed self-assembled monolayer of thiolated hydroxynaphthoquinone and thiolated oligonucleotide. Electrochemical changes resulting from hybridization were evidenced with oligonucleotide targets (as models...

  11. Multimodal label-free microscopy

    Directory of Open Access Journals (Sweden)

    Nicolas Pavillon

    2014-09-01

    Full Text Available This paper reviews the different multimodal applications based on a large extent of label-free imaging modalities, ranging from linear to nonlinear optics, while also including spectroscopic measurements. We put specific emphasis on multimodal measurements going across the usual boundaries between imaging modalities, whereas most multimodal platforms combine techniques based on similar light interactions or similar hardware implementations. In this review, we limit the scope to focus on applications for biology such as live cells or tissues, since by their nature of being alive or fragile, we are often not free to take liberties with the image acquisition times and are forced to gather the maximum amount of information possible at one time. For such samples, imaging by a given label-free method usually presents a challenge in obtaining sufficient optical signal or is limited in terms of the types of observable targets. Multimodal imaging is then particularly attractive for these samples in order to maximize the amount of measured information. While multimodal imaging is always useful in the sense of acquiring additional information from additional modes, at times it is possible to attain information that could not be discovered using any single mode alone, which is the essence of the progress that is possible using a multimodal approach.

  12. Label free fragment screening using surface plasmon resonance as a tool for fragment finding - analyzing parkin, a difficult CNS target.

    Directory of Open Access Journals (Sweden)

    Karin Regnström

    Full Text Available Surface Plasmon Resonance (SPR is rarely used as a primary High-throughput Screening (HTS tool in fragment-based approaches. With SPR instruments becoming increasingly high-throughput it is now possible to use SPR as a primary tool for fragment finding. SPR becomes, therefore, a valuable tool in the screening of difficult targets such as the ubiquitin E3 ligase Parkin. As a prerequisite for the screen, a large number of SPR tests were performed to characterize and validate the active form of Parkin. A set of compounds was designed and used to define optimal SPR assay conditions for this fragment screen. Using these conditions, more than 5000 pre-selected fragments from our in-house library were screened for binding to Parkin. Additionally, all fragments were simultaneously screened for binding to two off target proteins to exclude promiscuous binding compounds. A low hit rate was observed that is in line with hit rates usually obtained by other HTS screening assays. All hits were further tested in dose responses on the target protein by SPR for confirmation before channeling the hits into Nuclear Magnetic Resonance (NMR and other hit-confirmation assays.

  13. Development of Ionic Liquid Modified Disposable Graphite Electrodes for Label-Free Electrochemical Detection of DNA Hybridization Related to Microcystis spp.

    Directory of Open Access Journals (Sweden)

    Ceren Sengiz

    2015-09-01

    Full Text Available In this present study, ionic liquid (1-butyl-3-methylimidazolium hexafluorophosphate (IL modified pencil graphite electrode (IL-PGEs was developed for electrochemical monitoring of DNA hybridization related to Microcystis spp. (MYC. The characterization of IL-PGEs was performed using microscopic and electrochemical techniques. DNA hybridization related to MYC was then explored at the surface of IL-PGEs using differential pulse voltammetry (DPV technique. After the experimental parameters were optimized, the sequence-selective DNA hybridization related to MYC was performed in the case of hybridization between MYC probe and its complementary DNA target, noncomplementary (NC or mismatched DNA sequence (MM, or and in the presence of mixture of DNA target: NC (1:1 and DNA target: MM (1:1.

  14. Polypyrrole-poly(3,4-ethylenedioxythiophene)-Ag (PPy-PEDOT-Ag) nanocomposite films for label-free electrochemical DNA sensing.

    Science.gov (United States)

    Radhakrishnan, S; Sumathi, C; Umar, Ahmad; Jae Kim, Sang; Wilson, J; Dharuman, V

    2013-09-15

    The electrochemical DNA hybridization sensing of bipolymer polypyrrole and poly(3,4-ethylenedioxythiophene) (PPy-PEDOT) nanotubes functionalized with Ag nanoparticles has been investigated. The bipolymer nanotubes are prepared by simple chemical route and silver nanoparticles (Ag) further deposited over the PPy-PEDOT nanotubes to form PPy-PEDOT-Ag nanocomposite films. DNA labeled at 5'end using 6-mercapto-1-hexhane (HS-ssDNA) is immobilized on the PPy-PEDOT-Ag surface to form PPy-PEDOT-Ag-S-ssDNA and hybridization sensing is done in phosphate buffer. The presence of Ag nanoparticles (~28±5nm) well dispersed in the polymer composite with high surface area, high electrical conductivity and catalytic activity provides desirable microenvironment for the immobilization of probe DNA with controlled orientation leading to increased hybridization efficiency with target DNA. The morphological and structural characterizations by a scanning electron microscope (SEM) and X-ray diffraction (XRD) confirm the nanotube structure of composite polymer while Raman measurements indicate the efficient interactions between the PPy, PEDOT, Ag and HS-ssDNA. The sensor effectively discriminates different target DNA sequences with PPy-PEDOT-Ag-S-ssDNA substrate. The observed dynamic detection range is found between 1×10(-11)M and 1×10(-14)M with the lowest detection limit (3 σ/b) of 5.4×10(-15)M. This observed value is of higher sensitivity than that for MWCNT-Ag, PANi-Au, MWCNT-PPy-Au and PPy-PANi-Au composites reported previously. Copyright © 2013 Elsevier B.V. All rights reserved.

  15. A label-free, fluorescence based assay for microarray

    Science.gov (United States)

    Niu, Sanjun

    DNA chip technology has drawn tremendous attention since it emerged in the mid 90's as a method that expedites gene sequencing by over 100-fold. DNA chip, also called DNA microarray, is a combinatorial technology in which different single-stranded DNA (ssDNA) molecules of known sequences are immobilized at specific spots. The immobilized ssDNA strands are called probes. In application, the chip is exposed to a solution containing ssDNA of unknown sequence, called targets, which are labeled with fluorescent dyes. Due to specific molecular recognition among the base pairs in the DNA, the binding or hybridization occurs only when the probe and target sequences are complementary. The nucleotide sequence of the target is determined by imaging the fluorescence from the spots. The uncertainty of background in signal detection and statistical error in data analysis, primarily due to the error in the DNA amplification process and statistical distribution of the tags in the target DNA, have become the fundamental barriers in bringing the technology into application for clinical diagnostics. Furthermore, the dye and tagging process are expensive, making the cost of DNA chips inhibitive for clinical testing. These limitations and challenges make it difficult to implement DNA chip methods as a diagnostic tool in a pathology laboratory. The objective of this dissertation research is to provide an alternative approach that will address the above challenges. In this research, a label-free assay is designed and studied. Polystyrene (PS), a commonly used polymeric material, serves as the fluorescence agent. Probe ssDNA is covalently immobilized on polystyrene thin film that is supported by a reflecting substrate. When this chip is exposed to excitation light, fluorescence light intensity from PS is detected as the signal. Since the optical constants and conformations of ssDNA and dsDNA (double stranded DNA) are different, the measured fluorescence from PS changes for the same

  16. Mapping the intracellular distribution of carbon nanotubes after targeted delivery to carcinoma cells using confocal Raman imaging as a label-free technique

    International Nuclear Information System (INIS)

    Lamprecht, C; Unterauer, B; Plochberger, B; Brameshuber, M; Hinterdorfer, P; Ebner, A; Gierlinger, N; Hild, S; Heister, E

    2012-01-01

    The uptake of carbon nanotubes (CNTs) by mammalian cells and their distribution within cells is being widely studied in recent years due to their increasing use for biomedical purposes. The two main imaging techniques used are confocal fluorescence microscopy and transmission electron microscopy (TEM). The former, however, requires labeling of the CNTs with fluorescent dyes, while the latter is a work-intensive technique that is unsuitable for in situ bio-imaging. Raman spectroscopy, on the other hand, presents a direct, straightforward and label-free alternative. Confocal Raman microscopy can be used to image the CNTs inside cells, exploiting the strong Raman signal connected to different vibrational modes of the nanotubes. In addition, cellular components, such as the endoplasmic reticulum and the nucleus, can be mapped. We first validate our method by showing that only when using the CNTs’ G band for intracellular mapping accurate results can be obtained, as mapping of the radial breathing mode (RBM) only shows a small fraction of CNTs. We then take a closer look at the exact localization of the nanotubes inside cells after folate receptor-mediated endocytosis and show that, after 8-10 h incubation, the majority of CNTs are localized around the nucleus. In summary, Raman imaging has enormous potential for imaging CNTs inside cells, which is yet to be fully realized. (paper)

  17. Simple and label-free electrochemical impedance Amelogenin gene hybridization biosensing based on reduced graphene oxide.

    Science.gov (United States)

    Benvidi, Ali; Rajabzadeh, Nooshin; Mazloum-Ardakani, Mohammad; Heidari, Mohammad Mehdi; Mulchandani, Ashok

    2014-08-15

    The increasing desire for sensitive, easy, low-cost, and label free methods for the detection of DNA sequences has become a vital matter in biomedical research. For the first time a novel label-free biosensor for sensitive detection of Amelogenin gene (AMEL) using reduced graphene oxide modified glassy carbon electrode (GCE/RGO) has been developed. In this work, detection of DNA hybridization of the target and probe DNA was investigated by electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). The optimum conditions were found for the immobilization of probe on RGO surface and its hybridization with the target DNA. CV and EIS carried out in an aqueous solution containing [Fe(CN)6](3-/4-) redox pair have been used for the biosensor characterization. The biosensor has a wide linear range from 1.0×10(-20) to 1.0×10(-14)M with the lower detection limit of 3.2×10(-21)M. Moreover, the present electrochemical detection offers some unique advantages such as ultrahigh sensitivity, simplicity, and feasibility for apparatus miniaturization in analytical tests. The excellent performance of the biosensor is attributed to large surface-to-volume ratio and high conductivity of RGO, which enhances the probe absorption and promotes direct electron transfer between probe and the electrode surface. This electrochemical DNA sensor could be used for the detection of specific ssDNA sequence in real biological samples. Copyright © 2014 Elsevier B.V. All rights reserved.

  18. PEG Functionalization of Whispering Gallery Mode Optical Microresonator Biosensors to Minimize Non-Specific Adsorption during Targeted, Label-Free Sensing

    Directory of Open Access Journals (Sweden)

    Fanyongjing Wang

    2015-07-01

    Full Text Available Whispering Gallery Mode (WGM optical microresonator biosensors are a powerful tool for targeted detection of analytes at extremely low concentrations. However, in complex environments, non-specific adsorption can significantly reduce their signal to noise ratio, limiting their accuracy. To overcome this, poly(ethylene glycol (PEG can be employed in conjunction with appropriate recognition elements to create a nonfouling surface capable of detecting targeted analytes. This paper investigates a general route for the addition of nonfouling elements to WGM optical biosensors to reduce non-specific adsorption, while also retaining high sensitivity. We use the avidin-biotin analyte-recognition element system, in conjunction with PEG nonfouling elements, as a proof-of-concept, and explore the extent of non-specific adsorption of lysozyme and fibrinogen at multiple concentrations, as well as the ability to detect avidin in a concentration-dependent fashion. Ellipsometry, contact angle measurement, fluorescence microscopy, and optical resonator characterization methods were used to study non-specific adsorption, the quality of the functionalized surface, and the biosensor’s performance. Using a recognition element ratio to nonfouling element ratio of 1:1, we showed that non-specific adsorption could be significantly reduced over the controls, and that high sensitivity could be maintained. Due to the frequent use of biotin-avidin-biotin sandwich complexes in functionalizing sensor surfaces with biotin-labeled recognition elements, this chemistry could provide a common basis for creating a non-fouling surface capable of targeted detection. This should improve the ability of WGM optical biosensors to operate in complex environments, extending their application towards real-world detection.

  19. Photonic Crystal Biosensor Chip for Label-Free Detection of Bacteria

    DEFF Research Database (Denmark)

    Kristensen, Martin; Krüger, Asger Christian; Groothoff, Nathaniel

    Narrow polarization-mixing resonances in planar photonic crystals are studied as candidate components for label-free refractive index sensors for detecting bacteria causing sepsis through the identification of DNA strands....

  20. Label-free probing of genes by time-domain terahertz sensing

    International Nuclear Information System (INIS)

    Bolivar, P Haring; Brucherseifer, M; Nagel, M; Kurz, H; Bosserhoff, A; Buettner, R

    2002-01-01

    A label-free sensing approach for the label-free characterization of genetic material with terahertz (THz) electromagnetic waves is presented. Time-resolved THz analysis of polynucleotides demonstrates a strong dependence of the complex refractive index of DNA molecules in the THz frequency range on their hybridization state. By monitoring THz signals one can thus infer the binding state (hybridized or denatured) of oligo- and polynucleotides, enabling the label-free determination the genetic composition of unknown DNA sequences. A broadband experimental proof-of-principle in a free-space analytic configuration, as well as a higher-sensitivity approach using integrated THz sensors reaching femtomol detection levels and demonstrating the capability to detect single-base mutations, are presented. The potential application for next generation high-throughput label-free genetic analytic systems is discussed

  1. Label-free probing of genes by time-domain terahertz sensing

    Energy Technology Data Exchange (ETDEWEB)

    Bolivar, P Haring [Institut fuer Halbleitertechnik, RWTH Aachen, Sommerfeldstr. 24, D-52056 Aachen (Germany); Brucherseifer, M [Institut fuer Halbleitertechnik, RWTH Aachen, Sommerfeldstr. 24, D-52056 Aachen (Germany); Nagel, M [Institut fuer Halbleitertechnik, RWTH Aachen, Sommerfeldstr. 24, D-52056 Aachen (Germany); Kurz, H [Institut fuer Halbleitertechnik, RWTH Aachen, Sommerfeldstr. 24, D-52056 Aachen (Germany); Bosserhoff, A [Institut fuer Pathologie, Universitaet Regensburg, Franz-Josef-Strauss-Allee 11, D-93053 Regensburg (Germany); Buettner, R [Institut fuer Pathologie, Universitaetsklinikum Bonn, Sigmund-Freud-Str. 25, D-53127 Bonn (Germany)

    2002-11-07

    A label-free sensing approach for the label-free characterization of genetic material with terahertz (THz) electromagnetic waves is presented. Time-resolved THz analysis of polynucleotides demonstrates a strong dependence of the complex refractive index of DNA molecules in the THz frequency range on their hybridization state. By monitoring THz signals one can thus infer the binding state (hybridized or denatured) of oligo- and polynucleotides, enabling the label-free determination the genetic composition of unknown DNA sequences. A broadband experimental proof-of-principle in a free-space analytic configuration, as well as a higher-sensitivity approach using integrated THz sensors reaching femtomol detection levels and demonstrating the capability to detect single-base mutations, are presented. The potential application for next generation high-throughput label-free genetic analytic systems is discussed.

  2. A Label-Free Photoluminescence Genosensor Using Nanostructured Magnesium Oxide for Cholera Detection

    Science.gov (United States)

    Patel, Manoj Kumar; Ali, Md. Azahar; Krishnan, Sadagopan; Agrawal, Ved Varun; Al Kheraif, Abdulaziz A.; Fouad, H.; Ansari, Z. A.; Ansari, S. G.; Malhotra, Bansi D.

    2015-11-01

    Nanomaterial-based photoluminescence (PL) diagnostic devices offer fast and highly sensitive detection of pesticides, DNA, and toxic agents. Here we report a label-free PL genosensor for sensitive detection of Vibrio cholerae that is based on a DNA hybridization strategy utilizing nanostructured magnesium oxide (nMgO; size >30 nm) particles. The morphology and size of the synthesized nMgO were determined by transmission electron microscopic (TEM) studies. The probe DNA (pDNA) was conjugated with nMgO and characterized by X-ray photoelectron and Fourier transform infrared spectroscopic techniques. The target complementary genomic DNA (cDNA) isolated from clinical samples of V. cholerae was subjected to DNA hybridization studies using the pDNA-nMgO complex and detection of the cDNA was accomplished by measuring changes in PL intensity. The PL peak intensity measured at 700 nm (red emission) increases with the increase in cDNA concentration. A linear range of response in the developed PL genosensor was observed from 100 to 500 ng/μL with a sensitivity of 1.306 emi/ng, detection limit of 3.133 ng/μL and a regression coefficient (R2) of 0.987. These results show that this ultrasensitive PL genosensor has the potential for applications in the clinical diagnosis of cholera.

  3. Label-free logic modules and two-layer cascade based on stem-loop probes containing a G-quadruplex domain.

    Science.gov (United States)

    Guo, Yahui; Cheng, Junjie; Wang, Jine; Zhou, Xiaodong; Hu, Jiming; Pei, Renjun

    2014-09-01

    A simple, versatile, and label-free DNA computing strategy was designed by using toehold-mediated strand displacement and stem-loop probes. A full set of logic gates (YES, NOT, OR, NAND, AND, INHIBIT, NOR, XOR, XNOR) and a two-layer logic cascade were constructed. The probes contain a G-quadruplex domain, which was blocked or unfolded through inputs initiating strand displacement and the obviously distinguishable light-up fluorescent signal of G-quadruplex/NMM complex was used as the output readout. The inputs are the disease-specific nucleotide sequences with potential for clinic diagnosis. The developed versatile computing system based on our label-free and modular strategy might be adapted in multi-target diagnosis through DNA hybridization and aptamer-target interaction. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Modeling the microscopic electrical properties of thrombin binding aptamer (TBA) for label-free biosensors

    Science.gov (United States)

    Alfinito, Eleonora; Reggiani, Lino; Cataldo, Rosella; De Nunzio, Giorgio; Giotta, Livia; Guascito, Maria Rachele

    2017-02-01

    Aptamers are chemically produced oligonucleotides, able to bind a variety of targets such as drugs, proteins and pathogens with high sensitivity and selectivity. Therefore, aptamers are largely employed for producing label-free biosensors (aptasensors), with significant applications in diagnostics and drug delivery. In particular, the anti-thrombin aptamers are biomolecules of high interest for clinical use, because of their ability to recognize and bind the thrombin enzyme. Among them, the DNA 15-mer aptamer (TBA), has been widely explored around the possibility of using it in aptasensors. This paper proposes a microscopic model of the electrical properties of TBA and of the aptamer-thrombin complex, combining information from both structure and function, following the issues addressed in an emerging branch of electronics known as proteotronics. The theoretical results are compared and validated with measurements reported in the literature. Finally, the model suggests resistance measurements as a novel tool for testing aptamer-target affinity.

  5. A cascade autocatalytic strand displacement amplification and hybridization chain reaction event for label-free and ultrasensitive electrochemical nucleic acid biosensing.

    Science.gov (United States)

    Chen, Zhiqiang; Liu, Ying; Xin, Chen; Zhao, Jikuan; Liu, Shufeng

    2018-04-23

    Herein, an autocatalytic strand displacement amplification (ASDA) strategy was proposed for the first time, which was further ingeniously coupled with hybridization chain reaction (HCR) event for the isothermal, label-free and multiple amplification toward nucleic acid detection. During the ASDA module, the target recognition opens the immobilized hairpin probe (IP) and initiates the annealing of the auxiliary DNA strand (AS) with the opened IP for the successive polymerization and nicking reaction in the presence of DNA polymerase and nicking endonuclease. This induces the target recycling and generation of a large amount of intermediate DNA sequences, which can be used as target analogy to execute the autocatalytic strand displacement amplification. Simultaneously, the introduced AS strand can propagate the HCR between two hairpins (H1 and H2) to form a linear DNA concatamer with cytosine (C)-rich loop region, which can facilitate the in-situ synthesis of silver nanoclusters (AgNCs) as electrochemical tags for further amplification toward target responses. With current cascade ASDA and HCR strategy, the detection of target DNA could be achieved with a low detection limit of about 0.16 fM and a good selectivity. The developed biosensor also exhibits the distinct advantages of flexibility and simplicity in probe design and biosensor fabrication, and label-free electrochemical detection, thus opens a promising avenue for the detection of nucleic acid with low abundance in bioanalysis and clinical biomedicine. Copyright © 2018 Elsevier B.V. All rights reserved.

  6. A G-quadruplex-based Label-free Fluorometric Aptasensor for Adenosine Triphosphate Detection.

    Science.gov (United States)

    Li, Li Juan; Tian, Xue; Kong, Xiang Juan; Chu, Xia

    2015-01-01

    A G-quadruplex-based, label-free fluorescence assay was demonstrated for the detection of adenosine triphosphate (ATP). A double-stranded DNA (dsDNA), hybridized by ATP-aptamer and its complementary sequence, was employed as a substrate for ATP binding. SYBR Green I (SG I) was a fluorescent probe and exonuclease III (Exo III) was a nuclease to digest the dsDNA. Consequently, in the absence of ATP, the dsDNA was inset with SG I and was digested by Exo III, resulting in a low background signal. In the presence of ATP, the aptamer in dsDNA folded into a G-quadruplex structure that resisted the digestion of Exo III. SG I was inserted into the structure, showing high fluorescence. Owing to a decrease of the background noise, a high signal-to-noise ratio could be obtained. This sensor can detect ATP with a concentration ranging from 50 μM to 5 mM, and possesses a capacity for the sensitive determination of other targets.

  7. Label-Free Raman Imaging to Monitor Breast Tumor Signatures

    Science.gov (United States)

    Ciubuc, John

    Methods built on Raman spectroscopy have shown major potential in describing and discriminating between malignant and benign specimens. Accurate, real-time medical diagnosis benefits in substantial improvements through this vibrational optical method. Not only is acquisition of data possible in milliseconds and analysis in minutes, Raman allows concurrent detection and monitoring of all biological components. Besides validating a significant Raman signature distinction between non-tumorigenic (MCF-10A) and tumorigenic (MCF-7) breast epithelial cells, this study reveals a label-free method to assess overexpression of epidermal growth factor receptors (EGFR) in tumor cells. EGFR overexpression sires Raman features associated with phosphorylated threonine and serine, and modifications of DNA/RNA characteristics. Investigations by gel electrophoresis reveal EGF induction of phosphorylated Akt, agreeing with the Raman results. The analysis presented is a vital step toward Raman-based evaluation of EGF receptors in breast cancer cells. With the goal of clinically applying Raman-guided methods for diagnosis of breast tumors, the current results lay the basis for proving label-free optical alternatives in making prognosis of the disease.

  8. A label-free ultrasensitive fluorescence detection of viable Salmonella enteritidis using enzyme-induced cascade two-stage toehold strand-displacement-driven assembly of G-quadruplex DNA.

    Science.gov (United States)

    Zhang, Peng; Liu, Hui; Ma, Suzhen; Men, Shuai; Li, Qingzhou; Yang, Xin; Wang, Hongning; Zhang, Anyun

    2016-06-15

    The harm of Salmonella enteritidis (S. enteritidis ) to public health mainly by contaminating fresh food and water emphasizes the urgent need for rapid detection techniques to help control the spread of the pathogen. In this assay, an newly designed capture probe complex that contained specific S. enteritidis-aptamer and hybridized signal target sequence was used for viable S. enteritidis recognition directly. In the presence of the target S. enteritidis, single-stranded target sequences were liberated and initiated the replication-cleavage reaction, producing numerous G-quadruplex structures with a linker on the 3'-end. And then, the sensing system took innovative advantage of quadratic linker-induced strand-displacement for the first time to release target sequence in succession, leading to the cyclic reuse of the target sequences and cascade signal amplification, thereby achieving the successive production of G-quadruplex structures. The fluorescent dye, N-Methyl mesoporphyrin IX, binded to these G-quadruplex structures and generated significantly enhanced fluorescent signals to achieve highly sensitive detection of S. enteritidis down to 60 CFU/mL with a linear range from 10(2) to 10(7)CFU/mL. By coupling the cascade two-stage target sequences-recyclable toehold strand-displacement with aptamer-based target recognition successfully, it is the first report on a novel non-label, modification-free and DNA extraction-free ultrasensitive fluorescence biosensor for detecting viable S. enteritidis directly, which can discriminate from dead S. enteritidis. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. Progress of new label-free techniques for biosensors: a review.

    Science.gov (United States)

    Sang, Shengbo; Wang, Yajun; Feng, Qiliang; Wei, Ye; Ji, Jianlong; Zhang, Wendong

    2016-01-01

    The detection techniques used in biosensors can be broadly classified into label-based and label-free. Label-based detection relies on the specific properties of labels for detecting a particular target. In contrast, label-free detection is suitable for the target molecules that are not labeled or the screening of analytes which are not easy to tag. Also, more types of label-free biosensors have emerged with developments in biotechnology. The latest developed techniques in label-free biosensors, such as field-effect transistors-based biosensors including carbon nanotube field-effect transistor biosensors, graphene field-effect transistor biosensors and silicon nanowire field-effect transistor biosensors, magnetoelastic biosensors, optical-based biosensors, surface stress-based biosensors and other type of biosensors based on the nanotechnology are discussed. The sensing principles, configurations, sensing performance, applications, advantages and restriction of different label-free based biosensors are considered and discussed in this review. Most concepts included in this survey could certainly be applied to the development of this kind of biosensor in the future.

  10. A universal and label-free impedimetric biosensing platform for discrimination of single nucleotide substitutions in long nucleic acid strands.

    Science.gov (United States)

    Mills, Dawn M; Martin, Christopher P; Armas, Stephanie M; Calvo-Marzal, Percy; Kolpashchikov, Dmitry M; Chumbimuni-Torres, Karin Y

    2018-06-30

    We report a label-free universal biosensing platform for highly selective detection of long nucleic acid strands. The sensor consists of an electrode-immobilized universal stem-loop (USL) probe and two adaptor strands that form a 4J structure in the presence of a specific DNA/RNA analyte. The sensor was characterized by electrochemical impedance spectroscopy (EIS) using K 3 [Fe(CN) 6 ]/K 4 [Fe(CN) 6 ] redox couple in solution. An increase in charge transfer resistance (R CT ) was observed upon 4J structure formation, the value of which depends on the analyte length. Cyclic voltammetry (CV) was used to further characterize the sensor and monitor the electrochemical reaction in conjunction with thickness measurements of the mixed DNA monolayer obtained using spectroscopic ellipsometry. In addition, the electron transfer was calculated at the electrode/electrolyte interface using a rotating disk electrode. Limits of detection in the femtomolar range were achieved for nucleic acid targets of different lengths (22 nt, 60 nt, 200 nt). The sensor produced only a background signal in the presence of single base mismatched analytes, even in hundred times excess in concentration. This label-free and highly selective biosensing platform is versatile and can be used for universal detection of nucleic acids of varied lengths which could revolutionize point of care diagnostics for applications such as bacterial or cancer screening. Copyright © 2018 Elsevier B.V. All rights reserved.

  11. Label-free and substrate-free potentiometric aptasensing using polycation-sensitive membrane electrodes.

    Science.gov (United States)

    Ding, Jiawang; Chen, Yan; Wang, Xuewei; Qin, Wei

    2012-02-21

    A potentiometric label-free and substrate-free (LFSF) aptasensing strategy which eliminates the labeling, separation, and immobilization steps is described in this paper. An aptamer binds specifically to a target molecule via reaction incubation, which could induce a change in the aptamer conformation from a random coil-like configuration to a rigid folded structure. Such a target binding-induced aptamer conformational change effectively prevents the aptamer from electrostatically interacting with the protamine binding domain. This could either shift the response curve for the potentiometric titration of the aptamer with protamine as monitored by a conventional polycation-sensitive membrane electrode or change the current-dependent potential detected by a protamine-conditioned polycation-sensitive electrode with the pulsed current-driven ion fluxes of protamine across the polymeric membrane. Using adenosine triphosphate (ATP) as a model analyte, the proposed concept offers potentiometric detection of ATP down to the submicromolar concentration range and has been applied to the determination of ATP in HeLa cells. In contrast to the current LFSF aptasensors based on optical detection, the proposed strategy allows the LFSF biosensing of aptamer/target binding events in a homogeneous solution via electrochemical transduction. It is anticipated that the proposed strategy will lay a foundation for development of potentiometric sensors for LFSF aptasensing of a variety of analytes where target binding-induced conformational changes such as the formation of folded structures and the opening of DNA hairpin loops are involved.

  12. Label-Free Raman Imaging to Monitor Breast Tumor Signatures.

    Science.gov (United States)

    Manciu, Felicia S; Ciubuc, John D; Parra, Karla; Manciu, Marian; Bennet, Kevin E; Valenzuela, Paloma; Sundin, Emma M; Durrer, William G; Reza, Luis; Francia, Giulio

    2017-08-01

    Although not yet ready for clinical application, methods based on Raman spectroscopy have shown significant potential in identifying, characterizing, and discriminating between noncancerous and cancerous specimens. Real-time and accurate medical diagnosis achievable through this vibrational optical method largely benefits from improvements in current technological and software capabilities. Not only is the acquisition of spectral information now possible in milliseconds and analysis of hundreds of thousands of data points achieved in minutes, but Raman spectroscopy also allows simultaneous detection and monitoring of several biological components. Besides demonstrating a significant Raman signature distinction between nontumorigenic (MCF-10A) and tumorigenic (MCF-7) breast epithelial cells, our study demonstrates that Raman can be used as a label-free method to evaluate epidermal growth factor activity in tumor cells. Comparative Raman profiles and images of specimens in the presence or absence of epidermal growth factor show important differences in regions attributed to lipid, protein, and nucleic acid vibrations. The occurrence, which is dependent on the presence of epidermal growth factor, of new Raman features associated with the appearance of phosphothreonine and phosphoserine residues reflects a signal transduction from the membrane to the nucleus, with concomitant modification of DNA/RNA structural characteristics. Parallel Western blotting analysis reveals an epidermal growth factor induction of phosphorylated Akt protein, corroborating the Raman results. The analysis presented in this work is an important step toward Raman-based evaluation of biological activity of epidermal growth factor receptors on the surfaces of breast cancer cells. With the ultimate future goal of clinically implementing Raman-guided techniques for the diagnosis of breast tumors (e.g., with regard to specific receptor activity), the current results just lay the foundation for

  13. Label-free cell-cycle analysis by high-throughput quantitative phase time-stretch imaging flow cytometry

    Science.gov (United States)

    Mok, Aaron T. Y.; Lee, Kelvin C. M.; Wong, Kenneth K. Y.; Tsia, Kevin K.

    2018-02-01

    Biophysical properties of cells could complement and correlate biochemical markers to characterize a multitude of cellular states. Changes in cell size, dry mass and subcellular morphology, for instance, are relevant to cell-cycle progression which is prevalently evaluated by DNA-targeted fluorescence measurements. Quantitative-phase microscopy (QPM) is among the effective biophysical phenotyping tools that can quantify cell sizes and sub-cellular dry mass density distribution of single cells at high spatial resolution. However, limited camera frame rate and thus imaging throughput makes QPM incompatible with high-throughput flow cytometry - a gold standard in multiparametric cell-based assay. Here we present a high-throughput approach for label-free analysis of cell cycle based on quantitative-phase time-stretch imaging flow cytometry at a throughput of > 10,000 cells/s. Our time-stretch QPM system enables sub-cellular resolution even at high speed, allowing us to extract a multitude (at least 24) of single-cell biophysical phenotypes (from both amplitude and phase images). Those phenotypes can be combined to track cell-cycle progression based on a t-distributed stochastic neighbor embedding (t-SNE) algorithm. Using multivariate analysis of variance (MANOVA) discriminant analysis, cell-cycle phases can also be predicted label-free with high accuracy at >90% in G1 and G2 phase, and >80% in S phase. We anticipate that high throughput label-free cell cycle characterization could open new approaches for large-scale single-cell analysis, bringing new mechanistic insights into complex biological processes including diseases pathogenesis.

  14. Label-free fluorescence strategy for sensitive microRNA detection based on isothermal exponential amplification and graphene oxide.

    Science.gov (United States)

    Li, Wei; Hou, Ting; Wu, Min; Li, Feng

    2016-01-01

    MicroRNAs (miRNAs) play an important role in many biological processes, and have been regarded as potential targets and biomarkers in cancer diagnosis and therapy. Also, to meet the big challenge imposed by the characteristics of miRNAs, such as small size and vulnerability to enzymatic digestion, it is of great importance to develop accurate, sensitive and simple miRNA assays. Herein, we developed a label-free fluorescence strategy for sensitive miRNA detection by combining isothermal exponential amplification and the unique features of SYBR Green I (SG) and graphene oxide (GO), in which SG gives significantly enhanced fluorescence upon intercalation into double-stranded DNAs (dsDNAs), and GO selectively adsorbs miRNA, single-stranded DNA and SG, to protect miRNA from enzymatic digestion, and to quench the fluorescence of the adsorbed SG. In the presence of the target miRNA, the ingeniously designed hairpin probe (HP) is unfolded and the subsequent polymerization and strand displacement reaction takes place to initiate the target recycling process. The newly formed dsDNAs are then recognized and cleaved by the nicking enzyme, generating new DNA triggers with the same sequence as the target miRNA, which hybridize with intact HPs to initiate new extension reactions. As a result, the circular exponential amplification for target miRNA is achieved and large amount of dsDNAs are formed to generate significantly enhanced fluorescence upon the intercalation of SG. Thus sensitive and selective fluorescence miRNA detection is realized, and the detection limit of 3 fM is obtained. Besides, this method exhibits additional advantages of simplicity and low cost, since expensive and tedious labeling process is avoided. Therefore, the as-proposed label-free fluorescence strategy has great potential in the applications in miRNA-related clinical practices and biochemical researches. Copyright © 2015 Elsevier B.V. All rights reserved.

  15. Label-free single-cell separation and imaging of cancer cells using an integrated microfluidic system

    DEFF Research Database (Denmark)

    Antfolk, Maria; Kim, Soo Hyeon; Koizumi, Saori

    2017-01-01

    , an integrated system is presented that efficiently eliminates this risk by integrating label-free separation with single cell arraying of the target cell population, enabling direct on-chip tumor cell identification and enumeration. Prostate cancer cells (DU145) spiked into a sample with whole blood...... a fully integrated system for rapid label-free separation and on-chip phenotypic characterization of circulating tumor cells from peripheral venous blood in clinical practice....

  16. An aggregated perylene-based broad-spectrum, efficient and label-free quencher for multiplexed fluorescent bioassays.

    Science.gov (United States)

    Liu, Tao; Hu, Rong; Lv, Yi-Fan; Wu, Yuan; Liang, Hao; Huan, Shuang-Yan; Zhang, Xiao-Bing; Tan, Weihong; Yu, Ru-Qin

    2014-08-15

    Fluorescent sensing systems based on the quenching of fluorophores have found wide applications in bioassays. An efficient quencher will endow the sensing system a high sensitivity. The frequently used quenchers are based on organic molecules or nanomaterials, which usually need tedious synthesizing and modifying steps, and exhibit different quenching efficiencies to different fluorophores. In this work, we for the first time report that aggregated perylene derivative can serve as a broad-spectrum and label-free quencher that is able to efficiently quench a variety of fluorophores, such as green, red and far red dyes labeled on DNA. By choosing nucleases as model biomolecules, such a broad-spectrum quencher was then employed to construct a multiplexed bioassay platform through a label-free manner. Due to the high quenching efficiency of the aggregated perylene, the proposed platform could detect nuclease with high sensitivity, with a detection limit of 0.03U/mL for EcoRV, and 0.05U/mL for EcoRI. The perylene quencher does not affect the activity of nuclease, which makes it possible to design post-addition type bioassay platform. Moreover, the proposed platform allows simultaneous and multicolor analysis of nucleases in homogeneous solution, demonstrating its value of potential application in rapid screening of multiple bio-targets. Copyright © 2014 Elsevier B.V. All rights reserved.

  17. Label-free detection of sex determining region Y (SRY) via capacitive biosensor

    KAUST Repository

    Sivashankar, Shilpa

    2016-10-20

    In this work, we present for the first time, the use of a simple fractal capacitive biosensor for the quantification and detection of sex-determining region Y (SRY) genes. This section of genetic code, which is found on the Y chromosome, finds importance for study as it causes fetuses to develop characteristics of male sex-like gonads when a mutation occurs. It is also an important genetic code in men, and disorders involving the SRY gene can cause infertility and sexual malfunction that lead to a variety of gene mutational disorders. We have therefore designed silicon-based, label-free fractal capacitive biosensors to quantify various proteins and genes. We take advantage of a good dielectric material, Parylene C for enhancing the performance of the sensors. We have integrated these sensors with a simple microchannel for easy handling of fluids on the detection area. The read-out value of an Agilent LCR meter used to measure capacitance of the sensor at a frequency of 1 MHz determined gene specificity and gene quantification. These data revealed that the capacitance measurement of the capacitive biosensor for the SRY gene depended on both the target and the concentration of DNA. The experimental outcomes in the present study can be used to detect DNA and its variations in crucial fields that have a great impact on our daily lives, such as clinical and veterinary diagnostics, industrial and environmental testing and forensic sciences.

  18. Single-walled carbon nanotubes based chemiresistive genosensor for label-free detection of human rheumatic heart disease

    Science.gov (United States)

    Singh, Swati; Kumar, Ashok; Khare, Shashi; Mulchandani, Ashok; Rajesh

    2014-11-01

    A specific and ultrasensitive, label free single-walled carbon nanotubes (SWNTs) based chemiresistive genosensor was fabricated for the early detection of Streptococcus pyogenes infection in human causing rheumatic heart disease. The mga gene of S. pyogenes specific 24 mer ssDNA probe was covalently immobilized on SWNT through a molecular bilinker, 1-pyrenemethylamine, using carbodiimide coupling reaction. The sensor was characterized by the current-voltage (I-V) characteristic curve and scanning electron microscopy. The sensing performance of the sensor was studied with respect to changes in conductance in SWNT channel based on hybridization of the target S. pyogenes single stranded genomic DNA (ssG-DNA) to its complementary 24 mer ssDNA probe. The sensor shows negligible response to non-complementary Staphylococcus aureus ssG-DNA, confirming the specificity of the sensor only with S. pyogenes. The genosensor exhibited a linear response to S. pyogenes G-DNA from 1 to1000 ng ml-1 with a limit of detection of 0.16 ng ml-1.

  19. Single-walled carbon nanotubes based chemiresistive genosensor for label-free detection of human rheumatic heart disease

    International Nuclear Information System (INIS)

    Singh, Swati; Kumar, Ashok; Khare, Shashi; Mulchandani, Ashok; Rajesh

    2014-01-01

    A specific and ultrasensitive, label free single-walled carbon nanotubes (SWNTs) based chemiresistive genosensor was fabricated for the early detection of Streptococcus pyogenes infection in human causing rheumatic heart disease. The mga gene of S. pyogenes specific 24 mer ssDNA probe was covalently immobilized on SWNT through a molecular bilinker, 1-pyrenemethylamine, using carbodiimide coupling reaction. The sensor was characterized by the current-voltage (I-V) characteristic curve and scanning electron microscopy. The sensing performance of the sensor was studied with respect to changes in conductance in SWNT channel based on hybridization of the target S. pyogenes single stranded genomic DNA (ssG-DNA) to its complementary 24 mer ssDNA probe. The sensor shows negligible response to non-complementary Staphylococcus aureus ssG-DNA, confirming the specificity of the sensor only with S. pyogenes. The genosensor exhibited a linear response to S. pyogenes G-DNA from 1 to1000 ng ml −1 with a limit of detection of 0.16 ng ml −1

  20. Single-walled carbon nanotubes based chemiresistive genosensor for label-free detection of human rheumatic heart disease

    Energy Technology Data Exchange (ETDEWEB)

    Singh, Swati; Kumar, Ashok, E-mail: rajesh-csir@yahoo.com, E-mail: ashokigib@rediffmail.com [CSIR-Institute of Genomics and Integrative Biology, Mall Road, Delhi 110007 (India); Academy of Scientific and Innovative Research (AcSIR), New Delhi (India); Khare, Shashi [National Centre for Disease Control, Sham Nath Marg, Delhi 110054 (India); Mulchandani, Ashok [Department of Chemical and Environmental Engineering, University of California, Riverside, California 92521 (United States); Rajesh, E-mail: rajesh-csir@yahoo.com, E-mail: ashokigib@rediffmail.com [CSIR-National Physical Laboratory, Dr. K. S. Krishnan Road, New Delhi 110012 (India)

    2014-11-24

    A specific and ultrasensitive, label free single-walled carbon nanotubes (SWNTs) based chemiresistive genosensor was fabricated for the early detection of Streptococcus pyogenes infection in human causing rheumatic heart disease. The mga gene of S. pyogenes specific 24 mer ssDNA probe was covalently immobilized on SWNT through a molecular bilinker, 1-pyrenemethylamine, using carbodiimide coupling reaction. The sensor was characterized by the current-voltage (I-V) characteristic curve and scanning electron microscopy. The sensing performance of the sensor was studied with respect to changes in conductance in SWNT channel based on hybridization of the target S. pyogenes single stranded genomic DNA (ssG-DNA) to its complementary 24 mer ssDNA probe. The sensor shows negligible response to non-complementary Staphylococcus aureus ssG-DNA, confirming the specificity of the sensor only with S. pyogenes. The genosensor exhibited a linear response to S. pyogenes G-DNA from 1 to1000 ng ml{sup −1} with a limit of detection of 0.16 ng ml{sup −1}.

  1. Label-free genotyping of cytochrome P450 2D6*10 using ligation-mediated strand displacement amplification with DNAzyme-based chemiluminescence detection.

    Science.gov (United States)

    Wang, Hong-Qi; Wu, Zhan; Zhang, Yan; Tang, Li-Juan; Yu, Ru-Qin; Jiang, Jian-Hui

    2012-01-13

    Genotyping of cytochrome P450 monooxygenase 2D6*10 (CYP2D6*10) plays an important role in pharmacogenomics, especially in clinical drug therapy of Asian populations. This work reported a novel label-free technique for genotyping of CYP2D6*10 based on ligation-mediated strand displacement amplification (SDA) with DNAzyme-based chemiluminescence detection. Discrimination of single-base mismatch is firstly accomplished using DNA ligase to generate a ligation product. The ligated product then initiates a SDA reaction to produce aptamer sequences against hemin, which can be probed by chemiluminescence detection. The proposed strategy is used for the assay of CYP2D6*10 target and the genomic DNA. The results reveal that the proposed technique displays chemiluminescence responses in linear correlation to the concentrations of DNA target within the range from 1 pM to 1 nM. A detection limit of 0.1 pM and a signal-to-background ratio of 57 are achieved. Besides such high sensitivity, the proposed CYP2D6*10 genotyping strategy also offers superb selectivity, great robustness, low cost and simplified operations due to its label-free, homogeneous, and chemiluminescence-based detection format. These advantages suggest this technique may hold considerable potential for clinical CYP2D6*10 genotyping and association studies. Copyright © 2011 Elsevier B.V. All rights reserved.

  2. Phase sensitive spectral domain interferometry for label free biomolecular interaction analysis and biosensing applications

    Science.gov (United States)

    Chirvi, Sajal

    Biomolecular interaction analysis (BIA) plays vital role in wide variety of fields, which include biomedical research, pharmaceutical industry, medical diagnostics, and biotechnology industry. Study and quantification of interactions between natural biomolecules (proteins, enzymes, DNA) and artificially synthesized molecules (drugs) is routinely done using various labeled and label-free BIA techniques. Labeled BIA (Chemiluminescence, Fluorescence, Radioactive) techniques suffer from steric hindrance of labels on interaction site, difficulty of attaching labels to molecules, higher cost and time of assay development. Label free techniques with real time detection capabilities have demonstrated advantages over traditional labeled techniques. The gold standard for label free BIA is surface Plasmon resonance (SPR) that detects and quantifies the changes in refractive index of the ligand-analyte complex molecule with high sensitivity. Although SPR is a highly sensitive BIA technique, it requires custom-made sensor chips and is not well suited for highly multiplexed BIA required in high throughput applications. Moreover implementation of SPR on various biosensing platforms is limited. In this research work spectral domain phase sensitive interferometry (SD-PSI) has been developed for label-free BIA and biosensing applications to address limitations of SPR and other label free techniques. One distinct advantage of SD-PSI compared to other label-free techniques is that it does not require use of custom fabricated biosensor substrates. Laboratory grade, off-the-shelf glass or plastic substrates of suitable thickness with proper surface functionalization are used as biosensor chips. SD-PSI is tested on four separate BIA and biosensing platforms, which include multi-well plate, flow cell, fiber probe with integrated optics and fiber tip biosensor. Sensitivity of 33 ng/ml for anti-IgG is achieved using multi-well platform. Principle of coherence multiplexing for multi

  3. A Rapid Label-Free Fluorescent Aptasensor PicoGreen-Based Strategy for Aflatoxin B₁ Detection in Traditional Chinese Medicines.

    Science.gov (United States)

    Zhang, Cheng; Dou, Xiaowen; Zhang, Lei; Sun, Meifeng; Zhao, Ming; OuYang, Zhen; Kong, Dandan; Antonio, F Logrieco; Yang, Meihua

    2018-02-28

    Aflatoxin B₁ (AFB₁) is a very hazardous carcinogen, readily contaminating foodstuffs and traditional Chinese medicines (TCMs) that has inspired increasing health concerns due to dietary exposure. Colloidal nanocrystals have been proposed as optical labels for aptasensor assembly, but these typically require tedious multistep conjugation and suffer from unsatisfactory robustness when used for complex matrices. In the present study, we report a rapid and sensitive method for screening for trace AFB₁ levels in TCMs using a label-free fluorescent aptasensor PicoGreen dye-based strategy. Using PicoGreen to selectively measure complementary double-stranded DNA, fluorescence enhancement due to dsDNA is 'turned off' in the presence of AFB₁ due binding of aptamer target over complementary sequence. Self-assembly of a label-free fluorescent aptasensor based on AFB₁ aptamer and PicoGreen dye was performed. Due to competition between the complementary sequence and AFB₁ target, this rapid method was capable of highly sensitive and selective screening for AFB₁ in five types of TCMs. This proposed approach had a limit of detection as low as 0.1 μg·L -1 and good linearity with a range of 0.1-10 μg·L -1 (0.1-10 ppb). Among the 20 samples tested, 6 batches were found to be contaminated with AFB₁ using this method, which was confirmed using sophisticated liquid chromatography-electrospray ionization-tandem mass spectrometry/mass spectrometry analysis. The results of this study indicate the developed method has the potential to be a simple, quick, and sensitive tool for detecting AFB₁ in TCMs.

  4. Targeting DNA Replication Stress for Cancer Therapy

    Directory of Open Access Journals (Sweden)

    Jun Zhang

    2016-08-01

    Full Text Available The human cellular genome is under constant stress from extrinsic and intrinsic factors, which can lead to DNA damage and defective replication. In normal cells, DNA damage response (DDR mediated by various checkpoints will either activate the DNA repair system or induce cellular apoptosis/senescence, therefore maintaining overall genomic integrity. Cancer cells, however, due to constitutive growth signaling and defective DDR, may exhibit “replication stress” —a phenomenon unique to cancer cells that is described as the perturbation of error-free DNA replication and slow-down of DNA synthesis. Although replication stress has been proven to induce genomic instability and tumorigenesis, recent studies have counterintuitively shown that enhancing replicative stress through further loosening of the remaining checkpoints in cancer cells to induce their catastrophic failure of proliferation may provide an alternative therapeutic approach. In this review, we discuss the rationale to enhance replicative stress in cancer cells, past approaches using traditional radiation and chemotherapy, and emerging approaches targeting the signaling cascades induced by DNA damage. We also summarize current clinical trials exploring these strategies and propose future research directions including the use of combination therapies, and the identification of potential new targets and biomarkers to track and predict treatment responses to targeting DNA replication stress.

  5. Protein search for multiple targets on DNA

    Energy Technology Data Exchange (ETDEWEB)

    Lange, Martin [Johannes Gutenberg University, Mainz 55122 (Germany); Department of Chemistry, Rice University, Houston, Texas 77005 (United States); Kochugaeva, Maria [Department of Chemistry, Rice University, Houston, Texas 77005 (United States); Kolomeisky, Anatoly B., E-mail: tolya@rice.edu [Department of Chemistry, Rice University, Houston, Texas 77005 (United States); Center for Theoretical Biological Physics, Rice University, Houston, Texas 77005 (United States)

    2015-09-14

    Protein-DNA interactions are crucial for all biological processes. One of the most important fundamental aspects of these interactions is the process of protein searching and recognizing specific binding sites on DNA. A large number of experimental and theoretical investigations have been devoted to uncovering the molecular description of these phenomena, but many aspects of the mechanisms of protein search for the targets on DNA remain not well understood. One of the most intriguing problems is the role of multiple targets in protein search dynamics. Using a recently developed theoretical framework we analyze this question in detail. Our method is based on a discrete-state stochastic approach that takes into account most relevant physical-chemical processes and leads to fully analytical description of all dynamic properties. Specifically, systems with two and three targets have been explicitly investigated. It is found that multiple targets in most cases accelerate the search in comparison with a single target situation. However, the acceleration is not always proportional to the number of targets. Surprisingly, there are even situations when it takes longer to find one of the multiple targets in comparison with the single target. It depends on the spatial position of the targets, distances between them, average scanning lengths of protein molecules on DNA, and the total DNA lengths. Physical-chemical explanations of observed results are presented. Our predictions are compared with experimental observations as well as with results from a continuum theory for the protein search. Extensive Monte Carlo computer simulations fully support our theoretical calculations.

  6. Label-free all-electronic biosensing in microfluidic systems

    Science.gov (United States)

    Stanton, Michael A.

    Label-free, all-electronic detection techniques offer great promise for advancements in medical and biological analysis. Electrical sensing can be used to measure both interfacial and bulk impedance changes in conducting solutions. Electronic sensors produced using standard microfabrication processes are easily integrated into microfluidic systems. Combined with the sensitivity of radiofrequency electrical measurements, this approach offers significant advantages over competing biological sensing methods. Scalable fabrication methods also provide a means of bypassing the prohibitive costs and infrastructure associated with current technologies. We describe the design, development and use of a radiofrequency reflectometer integrated into a microfluidic system towards the specific detection of biologically relevant materials. We developed a detection protocol based on impedimetric changes caused by the binding of antibody/antigen pairs to the sensing region. Here we report the surface chemistry that forms the necessary capture mechanism. Gold-thiol binding was utilized to create an ordered alkane monolayer on the sensor surface. Exposed functional groups target the N-terminus, affixing a protein to the monolayer. The general applicability of this method lends itself to a wide variety of proteins. To demonstrate specificity, commercially available mouse anti- Streptococcus Pneumoniae monoclonal antibody was used to target the full-length recombinant pneumococcal surface protein A, type 2 strain D39 expressed by Streptococcus Pneumoniae. We demonstrate the RF response of the sensor to both the presence of the surface decoration and bound SPn cells in a 1x phosphate buffered saline solution. The combined microfluidic sensor represents a powerful platform for the analysis and detection of cells and biomolecules.

  7. Label and Label-Free Detection Techniques for Protein Microarrays

    Directory of Open Access Journals (Sweden)

    Amir Syahir

    2015-04-01

    Full Text Available Protein microarray technology has gone through numerous innovative developments in recent decades. In this review, we focus on the development of protein detection methods embedded in the technology. Early microarrays utilized useful chromophores and versatile biochemical techniques dominated by high-throughput illumination. Recently, the realization of label-free techniques has been greatly advanced by the combination of knowledge in material sciences, computational design and nanofabrication. These rapidly advancing techniques aim to provide data without the intervention of label molecules. Here, we present a brief overview of this remarkable innovation from the perspectives of label and label-free techniques in transducing nano‑biological events.

  8. Label-free and selective nonlinear fiber-optical biosensing

    DEFF Research Database (Denmark)

    Ott, Johan Raunkjær; Heuck, Mikkel; Agger, Christian

    2008-01-01

    We demonstrate that the inherent nonlinearity of a microstructured optical fiber (MOF) may be used to achieve label-free selective biosensing, thereby eliminating the need for post-processing of the fiber. This first nonlinear biosensor utilizes a change in the modulational instability (MI) gain...... for optimizing the sensitivity. The nonlinear sensor shows a sensitivity of around 10.4nm/nm, defined as the shift in resonance wavelength per nm biolayer, which is a factor of 7.5 higher than the hitherto only demonstrated label-free MOF biosensor....

  9. Label-free monitoring of diffusion in microfluidics

    DEFF Research Database (Denmark)

    Sørensen, Kristian Tølbøl; Kristensen, Anders

    2017-01-01

    Label-free, real-time detection of concentration gradients is demonstrated in a microfluidic H-filter, using an integrated photonic crystal slab sensor to monitor sample refractive index with spatial resolution. The recorded diffusion profiles reveal root-mean-square diffusion lengths for non...

  10. Label-free three-dimensional imaging of cell nucleus using third-harmonic generation microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Lin, Jian; Zheng, Wei; Wang, Zi; Huang, Zhiwei, E-mail: biehzw@nus.edu.sg [Optical Bioimaging Laboratory, Department of Biomedical Engineering, Faculty of Engineering, National University of Singapore, Singapore 117576 (Singapore)

    2014-09-08

    We report the implementation of the combined third-harmonic generation (THG) and two-photon excited fluorescence (TPEF) microscopy for label-free three-dimensional (3-D) imaging of cell nucleus morphological changes in liver tissue. THG imaging shows regular spherical shapes of normal hepatocytes nuclei with inner chromatin structures while revealing the condensation of chromatins and nuclear fragmentations in hepatocytes of diseased liver tissue. Colocalized THG and TPEF imaging provides complementary information of cell nuclei and cytoplasm in tissue. This work suggests that 3-D THG microscopy has the potential for quantitative analysis of nuclear morphology in cells at a submicron-resolution without the need for DNA staining.

  11. Label-free three-dimensional imaging of cell nucleus using third-harmonic generation microscopy

    International Nuclear Information System (INIS)

    Lin, Jian; Zheng, Wei; Wang, Zi; Huang, Zhiwei

    2014-01-01

    We report the implementation of the combined third-harmonic generation (THG) and two-photon excited fluorescence (TPEF) microscopy for label-free three-dimensional (3-D) imaging of cell nucleus morphological changes in liver tissue. THG imaging shows regular spherical shapes of normal hepatocytes nuclei with inner chromatin structures while revealing the condensation of chromatins and nuclear fragmentations in hepatocytes of diseased liver tissue. Colocalized THG and TPEF imaging provides complementary information of cell nuclei and cytoplasm in tissue. This work suggests that 3-D THG microscopy has the potential for quantitative analysis of nuclear morphology in cells at a submicron-resolution without the need for DNA staining.

  12. A label-free electrochemiluminescent sensor for ATP detection based on ATP-dependent ligation.

    Science.gov (United States)

    Zhao, Tingting; Lin, Chunshui; Yao, Qiuhong; Chen, Xi

    2016-07-01

    In this work, we describe a new label-free, sensitive and highly selective strategy for the electrochemiluminescent (ECL) detection of ATP at the picomolar level via ATP-induced ligation. The molecular-beacon like DNA probes (P12 complex) are self-assembled on a gold electrode. The presence of ATP leads to the ligation of P12 complex which blocks the digestion by Exonuclease III (Exo III). The protected P12 complex causes the intercalation of numerous ECL indicators (Ru(phen)3(2+)) into the duplex DNA grooves, resulting in significantly amplified ECL signal output. Since the ligating site of T4 DNA ligase and the nicking site of Exo III are the same, it involves no long time of incubation for conformation change. The proposed strategy combines the amplification power of enzyme and the inherent high sensitivity of the ECL technique and enables picomolar detection of ATP. The developed strategy also shows high selectivity against ATP analogs, which makes our new label-free and highly sensitive ligation-based method a useful addition to the amplified ATP detection arena. Copyright © 2016 Elsevier B.V. All rights reserved.

  13. Binary electrokinetic separation of target DNA from background DNA primers.

    Energy Technology Data Exchange (ETDEWEB)

    James, Conrad D.; Derzon, Mark Steven

    2005-10-01

    This report contains the summary of LDRD project 91312, titled ''Binary Electrokinetic Separation of Target DNA from Background DNA Primers''. This work is the first product of a collaboration with Columbia University and the Northeast BioDefense Center of Excellence. In conjunction with Ian Lipkin's lab, we are developing a technique to reduce false positive events, due to the detection of unhybridized reporter molecules, in a sensitive and multiplexed detection scheme for nucleic acids developed by the Lipkin lab. This is the most significant problem in the operation of their capability. As they are developing the tools for rapidly detecting the entire panel of hemorrhagic fevers this technology will immediately serve an important national need. The goal of this work was to attempt to separate nucleic acid from a preprocessed sample. We demonstrated the preconcentration of kilobase-pair length double-stranded DNA targets, and observed little preconcentration of 60 base-pair length single-stranded DNA probes. These objectives were accomplished in microdevice formats that are compatible with larger detection systems for sample pre-processing. Combined with Columbia's expertise, this technology would enable a unique, fast, and potentially compact method for detecting/identifying genetically-modified organisms and multiplexed rapid nucleic acid identification. Another competing approach is the DARPA funded IRIS Pharmaceutical TIGER platform which requires many hours for operation, and an 800k$ piece of equipment that fills a room. The Columbia/SNL system could provide a result in 30 minutes, at the cost of a few thousand dollars for the platform, and would be the size of a shoebox or smaller.

  14. 6. Label-free selective plane illumination microscopy of tissue samples

    Directory of Open Access Journals (Sweden)

    Muteb Alharbi

    2017-10-01

    Conclusion: Overall this method meets the demands of the current needs for 3D imaging tissue samples in a label-free manner. Label-free Selective Plane Microscopy directly provides excellent information about the structure of the tissue samples. This work has highlighted the superiority of Label-free Selective Plane Microscopy to current approaches to label-free 3D imaging of tissue.

  15. Hybridization chain reaction-based colorimetric aptasensor of adenosine 5'-triphosphate on unmodified gold nanoparticles and two label-free hairpin probes.

    Science.gov (United States)

    Gao, Zhuangqiang; Qiu, Zhenli; Lu, Minghua; Shu, Jian; Tang, Dianping

    2017-03-15

    This work designs a new label-free aptasensor for the colorimetric determination of small molecules (adenosine 5'-triphosphate, ATP) by using visible gold nanoparticles as the signal-generation tags, based on target-triggered hybridization chain reaction (HCR) between two hairpin DNA probes. The assay is carried out referring to the change in the color/absorbance by salt-induced aggregation of gold nanoparticles after the interaction with hairpins, gold nanoparticles and ATP. To construct such an assay system, two hairpin DNA probes with a short single-stranded DNA at the sticky end are utilized for interaction with gold nanoparticles. In the absence of target ATP, the hairpin DNA probes can prevent gold nanoparticles from the salt-induced aggregation through the interaction of the single-stranded DNA at the sticky end with gold nanoparticles. Upon target ATP introduction, the aptamer-based hairpin probe is opened to expose a new sticky end for the strand-displacement reaction with another complementary hairpin, thus resulting in the decreasing single-stranded DNA because of the consumption of hairpins. In this case, gold nanoparticles are uncovered owing to the formation of double-stranded DNA, which causes their aggregation upon addition of the salt, thereby leading to the change in the red-to-blue color. Under the optimal conditions, the HCR-based colorimetric assay presents good visible color or absorbance responses for the determination of target ATP at a concentration as low as 1.0nM. Importantly, the methodology can be further extended to quantitatively or qualitatively monitor other small molecules or biotoxins by changing the sequence of the corresponding aptamer. Copyright © 2016 Elsevier B.V. All rights reserved.

  16. Label-free optical biosensing with slot-waveguides.

    Science.gov (United States)

    Barrios, Carlos A; Bañuls, María José; González-Pedro, Victoria; Gylfason, Kristinn B; Sánchez, Benito; Griol, Amadeu; Maquieira, A; Sohlström, H; Holgado, M; Casquel, R

    2008-04-01

    We demonstrate label-free molecule detection by using an integrated biosensor based on a Si(3)N(4)/SiO(2) slot-waveguide microring resonator. Bovine serum albumin (BSA) and anti-BSA molecular binding events on the sensor surface are monitored through the measurement of resonant wavelength shifts with varying biomolecule concentrations. The biosensor exhibited sensitivities of 1.8 and 3.2 nm/(ng/mm(2)) for the detection of anti-BSA and BSA, respectively. The estimated detection limits are 28 and 16 pg/mm(2) for anti-BSA and BSA, respectively, limited by wavelength resolution.

  17. Responsive Hydrogels for Label-Free Signal Transduction within Biosensors

    Directory of Open Access Journals (Sweden)

    Kamila Gawel

    2010-04-01

    Full Text Available Hydrogels have found wide application in biosensors due to their versatile nature. This family of materials is applied in biosensing either to increase the loading capacity compared to two-dimensional surfaces, or to support biospecific hydrogel swelling occurring subsequent to specific recognition of an analyte. This review focuses on various principles underpinning the design of biospecific hydrogels acting through various molecular mechanisms in transducing the recognition event of label-free analytes. Towards this end, we describe several promising hydrogel systems that when combined with the appropriate readout platform and quantitative approach could lead to future real-life applications.

  18. Plasmonic nanoparticles-decorated diatomite biosilica: extending the horizon of on-chip chromatography and label-free biosensing.

    Science.gov (United States)

    Kong, Xianming; Li, Erwen; Squire, Kenny; Liu, Ye; Wu, Bo; Cheng, Li-Jing; Wang, Alan X

    2017-11-01

    Diatomite consists of fossilized remains of ancient diatoms and is a type of naturally abundant photonic crystal biosilica with multiple unique physical and chemical functionalities. In this paper, we explored the fluidic properties of diatomite as the matrix for on-chip chromatography and, simultaneously, the photonic crystal effects to enhance the plasmonic resonances of metallic nanoparticles for surface-enhanced Raman scattering (SERS) biosensing. The plasmonic nanoparticle-decorated diatomite biosilica provides a lab-on-a-chip capability to separate and detect small molecules from mixture samples with ultra-high detection sensitivity down to 1 ppm. We demonstrate the significant potential for biomedical applications by screening toxins in real biofluid, achieving simultaneous label-free biosensing of phenethylamine and miR21cDNA in human plasma with unprecedented sensitivity and specificity. To the best of our knowledge, this is the first time demonstration to detect target molecules from real biofluids by on-chip chromatography-SERS techniques. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Recognizing different tissues in human fetal femur cartilage by label-free Raman microspectroscopy

    Science.gov (United States)

    Kunstar, Aliz; Leijten, Jeroen; van Leuveren, Stefan; Hilderink, Janneke; Otto, Cees; van Blitterswijk, Clemens A.; Karperien, Marcel; van Apeldoorn, Aart A.

    2012-11-01

    Traditionally, the composition of bone and cartilage is determined by standard histological methods. We used Raman microscopy, which provides a molecular "fingerprint" of the investigated sample, to detect differences between the zones in human fetal femur cartilage without the need for additional staining or labeling. Raman area scans were made from the (pre)articular cartilage, resting, proliferative, and hypertrophic zones of growth plate and endochondral bone within human fetal femora. Multivariate data analysis was performed on Raman spectral datasets to construct cluster images with corresponding cluster averages. Cluster analysis resulted in detection of individual chondrocyte spectra that could be separated from cartilage extracellular matrix (ECM) spectra and was verified by comparing cluster images with intensity-based Raman images for the deoxyribonucleic acid/ribonucleic acid (DNA/RNA) band. Specific dendrograms were created using Ward's clustering method, and principal component analysis (PCA) was performed with the separated and averaged Raman spectra of cells and ECM of all measured zones. Overall (dis)similarities between measured zones were effectively visualized on the dendrograms and main spectral differences were revealed by PCA allowing for label-free detection of individual cartilaginous zones and for label-free evaluation of proper cartilaginous matrix formation for future tissue engineering and clinical purposes.

  20. Automated selected reaction monitoring software for accurate label-free protein quantification.

    Science.gov (United States)

    Teleman, Johan; Karlsson, Christofer; Waldemarson, Sofia; Hansson, Karin; James, Peter; Malmström, Johan; Levander, Fredrik

    2012-07-06

    Selected reaction monitoring (SRM) is a mass spectrometry method with documented ability to quantify proteins accurately and reproducibly using labeled reference peptides. However, the use of labeled reference peptides becomes impractical if large numbers of peptides are targeted and when high flexibility is desired when selecting peptides. We have developed a label-free quantitative SRM workflow that relies on a new automated algorithm, Anubis, for accurate peak detection. Anubis efficiently removes interfering signals from contaminating peptides to estimate the true signal of the targeted peptides. We evaluated the algorithm on a published multisite data set and achieved results in line with manual data analysis. In complex peptide mixtures from whole proteome digests of Streptococcus pyogenes we achieved a technical variability across the entire proteome abundance range of 6.5-19.2%, which was considerably below the total variation across biological samples. Our results show that the label-free SRM workflow with automated data analysis is feasible for large-scale biological studies, opening up new possibilities for quantitative proteomics and systems biology.

  1. DNA repair in cancer: emerging targets for personalized therapy

    International Nuclear Information System (INIS)

    Abbotts, Rachel; Thompson, Nicola; Madhusudan, Srinivasan

    2014-01-01

    Genomic deoxyribonucleic acid (DNA) is under constant threat from endogenous and exogenous DNA damaging agents. Mammalian cells have evolved highly conserved DNA repair machinery to process DNA damage and maintain genomic integrity. Impaired DNA repair is a major driver for carcinogenesis and could promote aggressive cancer biology. Interestingly, in established tumors, DNA repair activity is required to counteract oxidative DNA damage that is prevalent in the tumor microenvironment. Emerging clinical data provide compelling evidence that overexpression of DNA repair factors may have prognostic and predictive significance in patients. More recently, DNA repair inhibition has emerged as a promising target for anticancer therapy. Synthetic lethality exploits intergene relationships where the loss of function of either of two related genes is nonlethal, but loss of both causes cell death. Exploiting this approach by targeting DNA repair has emerged as a promising strategy for personalized cancer therapy. In the current review, we focus on recent advances with a particular focus on synthetic lethality targeting in cancer

  2. Label-free surface plasmon sensing towards cancer diagnostics

    Science.gov (United States)

    Sankaranarayanan, Goutham

    The main objective of this thesis is to develop a conventional, home-built SPR bio-sensor to demonstrate bio-sensing applications. This emphasizes the understanding of basic concepts of Surface Plasmon Resonance and various interrogation techniques. Intensity Modulation was opted to perform the label-free SPR bio-sensing experiments due to its cost-efficient and compact setup. Later, label-free surface plasmon sensing was carried out to study and understand the bio-molecular interactions between (1). BSA and Anti BSA molecules and (2). Exosome/Liposome on thin metal (Au) films. Exosomes are cell-derived vesicles present in bodily fluids like blood, saliva, urine, epididymal fluid containing miRNAs, RNA, proteins, etc., at stable quantities during normal health conditions. The exosomes comprise varied constituents based on their cell origin from where they are secreted and is specific to that particular origin. However an exacerbated release is observed during tumor or cancer conditions. This increased level of exosomes present in the sample, can be detected using the SPR bio-sensor demonstrated in this thesis and effective thickness of adsorption on Au surface can be estimated. Also, chemically synthesized liposome particles were studied to determine if they can generate an equivalent sensor response to that of exosomes to consider them as an alternate. Finally a 10ppb Mercury (Hg) sensing was performed as part of Environment Monitoring application and results have been tabulated and compared.

  3. Data from quantitative label free proteomics analysis of rat spleen

    Directory of Open Access Journals (Sweden)

    Khadar Dudekula

    2016-09-01

    Full Text Available The dataset presented in this work has been obtained using a label-free quantitative proteomic analysis of rat spleen. A robust method for extraction of proteins from rat spleen tissue and LC-MS-MS analysis was developed using a urea and SDS-based buffer. Different fractionation methods were compared. A total of 3484 different proteins were identified from the pool of all experiments run in this study (a total of 2460 proteins with at least two peptides. A total of 1822 proteins were identified from nine non-fractionated pulse gels, 2288 proteins and 2864 proteins were identified by SDS-PAGE fractionation into three and five fractions respectively. The proteomics data are deposited in ProteomeXchange Consortium via PRIDE PXD003520, Progenesis and Maxquant output are presented in the supported information. The generated list of proteins under different regimes of fractionation allow assessing the nature of the identified proteins; variability in the quantitative analysis associated with the different sampling strategy and allow defining a proper number of replicates for future quantitative analysis. Keywords: Spleen, Rat, Protein extraction, Label-free quantitative proteomics

  4. Label-free detection of breast masses using multiphoton microscopy.

    Directory of Open Access Journals (Sweden)

    Xiufeng Wu

    Full Text Available Histopathology forms the gold standard for the diagnosis of breast cancer. Multiphoton microscopy (MPM has been proposed to be a potentially powerful adjunct to current histopathological techniques. A label-free imaging based on two- photon excited fluorescence and second-harmonic generation is developed for differentiating normal breast tissues, benign, as well as breast cancer tissues. Human breast biopsies (including human normal breast tissues, benign as well as breast cancer tissues that are first imaged (fresh, unfixed, and unstained with MPM and are then processed for routine H-E histopathology. Our results suggest that the MPM images, obtained from these unprocessed biopsies, can readily distinguish between benign lesions and breast cancers. In the tissues of breast cancers, MPM showed that the tumor cells displayed marked cellular and nuclear pleomorphism. The tumor cells, characterized by irregular size and shape, enlarged nuclei, and increased nuclear-cytoplasmic ratio, infiltrated into disrupted connective tissue, leading to the loss of second-harmonic generation signals. For breast cancer, MPM diagnosis was 100% correct because the tissues of breast cancers did not have second-harmonic generation signals in MPM imaging. On the contrary, in benign breast masses, second-harmonic generation signals could be seen easily in MPM imaging. These observations indicate that MPM could be an important potential tool to provide label-free noninvasive diagnostic impressions that can guide surgeon in biopsy and patient management.

  5. Use of UV-vis-NIR spectroscopy to monitor label-free interaction between molecular recognition elements and erythropoietin on a gold-coated polycarbonate platform.

    Science.gov (United States)

    Citartan, Marimuthu; Gopinath, Subash C B; Tominaga, Junji; Chen, Yeng; Tang, Thean-Hock

    2014-08-01

    Label-free-based detection is pivotal for real-time monitoring of biomolecular interactions and to eliminate the need for labeling with tags that can occupy important binding sites of biomolecules. One simplest form of label-free-based detection is ultraviolet-visible-near-infrared (UV-vis-NIR) spectroscopy, which measure changes in reflectivity as a means to monitor immobilization and interaction of biomolecules with their corresponding partners. In biosensor development, the platform used for the biomolecular interaction should be suitable for different molecular recognition elements. In this study, gold (Au)-coated polycarbonate was used as a platform and as a proof-of-concept, erythropoietin (EPO), a doping substance widely abused by the athletes was used as the target. The interaction of EPO with its corresponding molecular recognition elements (anti-EPO monoclonal antibody and anti-EPO DNA aptamer) is monitored by UV-vis-NIR spectroscopy. Prior to this, to show that UV-vis-NIR spectroscopy is a suitable method for measuring biomolecular interaction, the interaction between biotin and streptavidin was demonstrated via this strategy and reflectivity of this interaction decreased by 25%. Subsequent to this, interaction of the EPO with anti-EPO monoclonal antibody and anti-EPO DNA aptamer resulted in the decrease of reflectivity by 5% and 10%, respectively. The results indicated that Au-coated polycarbonate could be an ideal biosensor platform for monitoring biomolecular interactions using UV-vis-NIR spectroscopy. A smaller version of the Au-coated polycarbonate substrates can be derived from the recent set-up, to be applied towards detecting EPO abuse among atheletes. Copyright © 2014 Elsevier B.V. All rights reserved.

  6. Real-Time Label-Free Direct Electronic Monitoring of Topoisomerase Enzyme Binding Kinetics on Graphene.

    Science.gov (United States)

    Zuccaro, Laura; Tesauro, Cinzia; Kurkina, Tetiana; Fiorani, Paola; Yu, Hak Ki; Knudsen, Birgitta R; Kern, Klaus; Desideri, Alessandro; Balasubramanian, Kannan

    2015-11-24

    Monolayer graphene field-effect sensors operating in liquid have been widely deployed for detecting a range of analyte species often under equilibrium conditions. Here we report on the real-time detection of the binding kinetics of the essential human enzyme, topoisomerase I interacting with substrate molecules (DNA probes) that are immobilized electrochemically on to monolayer graphene strips. By monitoring the field-effect characteristics of the graphene biosensor in real-time during the enzyme-substrate interactions, we are able to decipher the surface binding constant for the cleavage reaction step of topoisomerase I activity in a label-free manner. Moreover, an appropriate design of the capture probes allows us to distinctly follow the cleavage step of topoisomerase I functioning in real-time down to picomolar concentrations. The presented results are promising for future rapid screening of drugs that are being evaluated for regulating enzyme activity.

  7. Hybrid integrated label-free chemical and biological sensors.

    Science.gov (United States)

    Mehrabani, Simin; Maker, Ashley J; Armani, Andrea M

    2014-03-26

    Label-free sensors based on electrical, mechanical and optical transduction methods have potential applications in numerous areas of society, ranging from healthcare to environmental monitoring. Initial research in the field focused on the development and optimization of various sensor platforms fabricated from a single material system, such as fiber-based optical sensors and silicon nanowire-based electrical sensors. However, more recent research efforts have explored designing sensors fabricated from multiple materials. For example, synthetic materials and/or biomaterials can also be added to the sensor to improve its response toward analytes of interest. By leveraging the properties of the different material systems, these hybrid sensing devices can have significantly improved performance over their single-material counterparts (better sensitivity, specificity, signal to noise, and/or detection limits). This review will briefly discuss some of the methods for creating these multi-material sensor platforms and the advances enabled by this design approach.

  8. Hybrid Integrated Label-Free Chemical and Biological Sensors

    Science.gov (United States)

    Mehrabani, Simin; Maker, Ashley J.; Armani, Andrea M.

    2014-01-01

    Label-free sensors based on electrical, mechanical and optical transduction methods have potential applications in numerous areas of society, ranging from healthcare to environmental monitoring. Initial research in the field focused on the development and optimization of various sensor platforms fabricated from a single material system, such as fiber-based optical sensors and silicon nanowire-based electrical sensors. However, more recent research efforts have explored designing sensors fabricated from multiple materials. For example, synthetic materials and/or biomaterials can also be added to the sensor to improve its response toward analytes of interest. By leveraging the properties of the different material systems, these hybrid sensing devices can have significantly improved performance over their single-material counterparts (better sensitivity, specificity, signal to noise, and/or detection limits). This review will briefly discuss some of the methods for creating these multi-material sensor platforms and the advances enabled by this design approach. PMID:24675757

  9. Nanophotonic label-free biosensors for environmental monitoring.

    Science.gov (United States)

    Chocarro-Ruiz, Blanca; Fernández-Gavela, Adrián; Herranz, Sonia; Lechuga, Laura M

    2017-06-01

    The field of environmental monitoring has experienced a substantial progress in the last years but still the on-site control of contaminants is an elusive problem. In addition, the growing number of pollutant sources is accompanied by an increasing need of having efficient early warning systems. Several years ago biosensor devices emerged as promising environmental monitoring tools, but their level of miniaturization and their fully operation outside the laboratory prevented their use on-site. In the last period, nanophotonic biosensors based on evanescent sensing have emerged as an outstanding choice for portable point-of-care diagnosis thanks to their capability, among others, of miniaturization, multiplexing, label-free detection and integration in lab-on-chip platforms. This review covers the most relevant nanophotonic biosensors which have been proposed (including interferometric waveguides, grating-couplers, microcavity resonators, photonic crystals and localized surface plasmon resonance sensors) and their recent application for environmental surveillance. Copyright © 2017 Elsevier Ltd. All rights reserved.

  10. Hybrid Integrated Label-Free Chemical and Biological Sensors

    Directory of Open Access Journals (Sweden)

    Simin Mehrabani

    2014-03-01

    Full Text Available Label-free sensors based on electrical, mechanical and optical transduction methods have potential applications in numerous areas of society, ranging from healthcare to environmental monitoring. Initial research in the field focused on the development and optimization of various sensor platforms fabricated from a single material system, such as fiber-based optical sensors and silicon nanowire-based electrical sensors. However, more recent research efforts have explored designing sensors fabricated from multiple materials. For example, synthetic materials and/or biomaterials can also be added to the sensor to improve its response toward analytes of interest. By leveraging the properties of the different material systems, these hybrid sensing devices can have significantly improved performance over their single-material counterparts (better sensitivity, specificity, signal to noise, and/or detection limits. This review will briefly discuss some of the methods for creating these multi-material sensor platforms and the advances enabled by this design approach.

  11. Signatures of DNA target selectivity by ETS transcription factors.

    Science.gov (United States)

    Poon, Gregory M K; Kim, Hye Mi

    2017-05-27

    The ETS family of transcription factors is a functionally heterogeneous group of gene regulators that share a structurally conserved, eponymous DNA-binding domain. DNA target specificity derives from combinatorial interactions with other proteins as well as intrinsic heterogeneity among ETS domains. Emerging evidence suggests molecular hydration as a fundamental feature that defines the intrinsic heterogeneity in DNA target selection and susceptibility to epigenetic DNA modification. This perspective invokes novel hypotheses in the regulation of ETS proteins in physiologic osmotic stress, their pioneering potential in heterochromatin, and the effects of passive and pharmacologic DNA demethylation on ETS regulation.

  12. Label free detection of 5′ hydroxymethylcytosine within CpG Islands using optical sensors

    Science.gov (United States)

    Hawk, Rasheeda M.; Armani, Andrea M.

    2014-01-01

    Significant research has been invested in correlating genetic variations with different disease probabilities. Recently, it has become apparent that other DNA modifications, such as the addition of a methyl or hydroxymethyl group to cytosine, can also play a role. While these modifications do not change the sequence, they can negatively impact the function. Therefore, it is critical to be able to both read the genetic code and identify these modifications. Currently, the detection of hydroxymethylated cytosine (5′hmC) and the two closely related variants, cytosine (C) and 5′methylcytosine (5′mC), relies on a combination of nucleotide modification steps, followed by PCR and gene sequencing. However, this approach is not ideal because transcription errors which are inherent to the PCR process can be misinterpreted as fluctuations in the relative C:5′mC:5′hmC concentrations. As such, an alternative method which does not rely on PCR or nucleotide modification is desirable. One approach is based on label-free optical resonant cavity sensors. In the present work, toroidal resonant cavity sensors are functionalized with antibodies to enable label-free detection and discrimination between C, 5′mC, and 5′hmC in real-time without PCR. Specifically, epoxide chemistry is used to covalently attach the 5′hmC antibody to the surface of the cavity. Subsequently, to thoroughly characterize the sensor platform, detection of C, 5′mC, and 5′hmC is performed over a concentration range from pM to nM. At low (pM) concentrations, the hydroxymethylated cytosine produces a significantly larger signal than the structurally similar epigenetic markers; thus demonstrating the applicability of this platform. PMID:25461158

  13. Dynamic and label-free high-throughput detection of biomolecular interactions based on phase-shift interferometry

    Science.gov (United States)

    Li, Qiang; Huang, Guoliang; Gan, Wupeng; Chen, Shengyi

    2009-08-01

    Biomolecular interactions can be detected by many established technologies such as fluorescence imaging, surface plasmon resonance (SPR)[1-4], interferometry and radioactive labeling of the analyte. In this study, we have designed and constructed a label-free, real-time sensing platform and its operating imaging instrument that detects interactions using optical phase differences from the accumulation of biological material on solid substrates. This system allows us to monitor biomolecular interactions in real time and quantify concentration changes during micro-mixing processes by measuring the changes of the optical path length (OPD). This simple interferometric technology monitors the optical phase difference resulting from accumulated biomolecular mass. A label-free protein chip that forms a 4×4 probe array was designed and fabricated using a commercial microarray robot spotter on solid substrates. Two positive control probe lines of BSA (Bovine Serum Albumin) and two experimental human IgG and goat IgG was used. The binding of multiple protein targets was performed and continuously detected by using this label-free and real-time sensing platform.

  14. Label-free screening of single biomolecules through resistive pulse sensing technology for precision medicine applications

    Science.gov (United States)

    Harrer, S.; Kim, S. C.; Schieber, C.; Kannam, S.; Gunn, N.; Moore, S.; Scott, D.; Bathgate, R.; Skafidas, S.; Wagner, J. M.

    2015-05-01

    Employing integrated nano- and microfluidic circuits for detecting and characterizing biological compounds through resistive pulse sensing technology is a vibrant area of research at the interface of biotechnology and nanotechnology. Resistive pulse sensing platforms can be customized to study virtually any particle of choice which can be threaded through a fluidic channel and enable label-free single-particle interrogation with the primary read-out signal being an electric current fingerprint. The ability to perform label-free molecular screening with single-molecule and even single binding site resolution makes resistive pulse sensing technology a powerful tool for analyzing the smallest units of biological systems and how they interact with each other on a molecular level. This task is at the core of experimental systems biology and in particular ‘omics research which in combination with next-generation DNA-sequencing and next-generation drug discovery and design forms the foundation of a novel disruptive medical paradigm commonly referred to as personalized medicine or precision medicine. DNA-sequencing has approached the 1000-Dollar-Genome milestone allowing for decoding a complete human genome with unmatched speed and at low cost. Increased sequencing efficiency yields massive amounts of genomic data. Analyzing this data in combination with medical and biometric health data eventually enables understanding the pathways from individual genes to physiological functions. Access to this information triggers fundamental questions for doctors and patients alike: what are the chances of an outbreak for a specific disease? Can individual risks be managed and if so how? Which drugs are available and how should they be applied? Could a new drug be tailored to an individual’s genetic predisposition fast and in an affordable way? In order to provide answers and real-life value to patients, the rapid evolvement of novel computing approaches for analyzing big data in

  15. Label-free screening of single biomolecules through resistive pulse sensing technology for precision medicine applications.

    Science.gov (United States)

    Harrer, S; Kim, S C; Schieber, C; Kannam, S; Gunn, N; Moore, S; Scott, D; Bathgate, R; Skafidas, S; Wagner, J M

    2015-05-08

    Employing integrated nano- and microfluidic circuits for detecting and characterizing biological compounds through resistive pulse sensing technology is a vibrant area of research at the interface of biotechnology and nanotechnology. Resistive pulse sensing platforms can be customized to study virtually any particle of choice which can be threaded through a fluidic channel and enable label-free single-particle interrogation with the primary read-out signal being an electric current fingerprint. The ability to perform label-free molecular screening with single-molecule and even single binding site resolution makes resistive pulse sensing technology a powerful tool for analyzing the smallest units of biological systems and how they interact with each other on a molecular level. This task is at the core of experimental systems biology and in particular 'omics research which in combination with next-generation DNA-sequencing and next-generation drug discovery and design forms the foundation of a novel disruptive medical paradigm commonly referred to as personalized medicine or precision medicine. DNA-sequencing has approached the 1000-Dollar-Genome milestone allowing for decoding a complete human genome with unmatched speed and at low cost. Increased sequencing efficiency yields massive amounts of genomic data. Analyzing this data in combination with medical and biometric health data eventually enables understanding the pathways from individual genes to physiological functions. Access to this information triggers fundamental questions for doctors and patients alike: what are the chances of an outbreak for a specific disease? Can individual risks be managed and if so how? Which drugs are available and how should they be applied? Could a new drug be tailored to an individual's genetic predisposition fast and in an affordable way? In order to provide answers and real-life value to patients, the rapid evolvement of novel computing approaches for analyzing big data in

  16. Data from quantitative label free proteomics analysis of rat spleen.

    Science.gov (United States)

    Dudekula, Khadar; Le Bihan, Thierry

    2016-09-01

    The dataset presented in this work has been obtained using a label-free quantitative proteomic analysis of rat spleen. A robust method for extraction of proteins from rat spleen tissue and LC-MS-MS analysis was developed using a urea and SDS-based buffer. Different fractionation methods were compared. A total of 3484 different proteins were identified from the pool of all experiments run in this study (a total of 2460 proteins with at least two peptides). A total of 1822 proteins were identified from nine non-fractionated pulse gels, 2288 proteins and 2864 proteins were identified by SDS-PAGE fractionation into three and five fractions respectively. The proteomics data are deposited in ProteomeXchange Consortium via PRIDE PXD003520, Progenesis and Maxquant output are presented in the supported information. The generated list of proteins under different regimes of fractionation allow assessing the nature of the identified proteins; variability in the quantitative analysis associated with the different sampling strategy and allow defining a proper number of replicates for future quantitative analysis.

  17. Label-free tissue scanner for colorectal cancer screening

    Science.gov (United States)

    Kandel, Mikhail E.; Sridharan, Shamira; Liang, Jon; Luo, Zelun; Han, Kevin; Macias, Virgilia; Shah, Anish; Patel, Roshan; Tangella, Krishnarao; Kajdacsy-Balla, Andre; Guzman, Grace; Popescu, Gabriel

    2017-06-01

    The current practice of surgical pathology relies on external contrast agents to reveal tissue architecture, which is then qualitatively examined by a trained pathologist. The diagnosis is based on the comparison with standardized empirical, qualitative assessments of limited objectivity. We propose an approach to pathology based on interferometric imaging of "unstained" biopsies, which provides unique capabilities for quantitative diagnosis and automation. We developed a label-free tissue scanner based on "quantitative phase imaging," which maps out optical path length at each point in the field of view and, thus, yields images that are sensitive to the "nanoscale" tissue architecture. Unlike analysis of stained tissue, which is qualitative in nature and affected by color balance, staining strength and imaging conditions, optical path length measurements are intrinsically quantitative, i.e., images can be compared across different instruments and clinical sites. These critical features allow us to automate the diagnosis process. We paired our interferometric optical system with highly parallelized, dedicated software algorithms for data acquisition, allowing us to image at a throughput comparable to that of commercial tissue scanners while maintaining the nanoscale sensitivity to morphology. Based on the measured phase information, we implemented software tools for autofocusing during imaging, as well as image archiving and data access. To illustrate the potential of our technology for large volume pathology screening, we established an "intrinsic marker" for colorectal disease that detects tissue with dysplasia or colorectal cancer and flags specific areas for further examination, potentially improving the efficiency of existing pathology workflows.

  18. Label-free in situ SERS imaging of biofilms.

    Science.gov (United States)

    Ivleva, Natalia P; Wagner, Michael; Szkola, Agathe; Horn, Harald; Niessner, Reinhard; Haisch, Christoph

    2010-08-12

    Surface-enhanced Raman scattering (SERS) is a promising technique for the chemical characterization of biological systems. It yields highly informative spectra, can be applied directly in aqueous environment, and has high sensitivity in comparison with normal Raman spectroscopy. Moreover, SERS imaging can provide chemical information with spatial resolution in the micrometer range (chemical imaging). In this paper, we report for the first time on the application of SERS for in situ, label-free imaging of biofilms and demonstrate the suitability of this technique for the characterization of the complex biomatrix. Biofilms, being communities of microorganisms embedded in a matrix of extracellular polymeric substances (EPS), represent the predominant mode of microbial life. Knowledge of the chemical composition and the structure of the biofilm matrix is important in different fields, e.g., medicine, biology, and industrial processes. We used colloidal silver nanoparticles for the in situ SERS analysis. Good SERS measurement reproducibility, along with a significant enhancement of Raman signals by SERS (>10(4)) and highly informative SERS signature, enables rapid SERS imaging (1 s for a single spectrum) of the biofilm matrix. Altogether, this work illustrates the potential of SERS for biofilm analysis, including the detection of different constituents and the determination of their distribution in a biofilm even at low biomass concentration.

  19. Label-free virus detection using silicon photonic microring resonators.

    Science.gov (United States)

    McClellan, Melinda S; Domier, Leslie L; Bailey, Ryan C

    2012-01-15

    Viruses represent a continual threat to humans through a number of mechanisms, which include disease, bioterrorism, and destruction of both plant and animal food resources. Many contemporary techniques used for the detection of viruses and viral infections suffer from limitations such as the need for extensive sample preparation or the lengthy window between infection and measurable immune response, for serological methods. In order to develop a method that is fast, cost-effective, and features reduced sample preparation compared to many other virus detection methods, we report the application of silicon photonic microring resonators for the direct, label-free detection of intact viruses in both purified samples as well as in a complex, real-world analytical matrix. As a model system, we demonstrate the quantitative detection of Bean pod mottle virus, a pathogen of great agricultural importance, with a limit of detection of 10 ng/mL. By simply grinding a small amount of leaf sample in buffer with a mortar and pestle, infected leaves can be identified over a healthy control with a total analysis time of less than 45 min. Given the inherent scalability and multiplexing capability of the semiconductor-based technology, we feel that silicon photonic microring resonators are well-positioned as a promising analytical tool for a number of viral detection applications. Copyright © 2011 Elsevier B.V. All rights reserved.

  20. A novel microfluidic chip electrophoresis strategy for simultaneous, label-free, multi-protein detection based on a graphene energy transfer biosensor.

    Science.gov (United States)

    Lin, Fengming; Zhao, Xiaochao; Wang, Jianshe; Yu, Shiyong; Deng, Yulin; Geng, Lina; Li, HuanJun

    2014-06-07

    A new type of high-throughput and parallel optical sensing platform with a single-color probe based on microfluidic chip electrophoresis combined with aptamer-carboxyfluorescein/graphene oxide energy transfer is reported here. Label-free protein multi-targets were detected, even in challenging complex samples without any pre-treatment.

  1. Engineering of magnetic DNA nanoparticles for tumor-targeted therapy

    International Nuclear Information System (INIS)

    Hosseinkhani, Hossein; Chen Yiru; He Wenjie; Hong Poda; Yu, Dah-Shyong; Domb, Abraham J.

    2013-01-01

    This study aims to engineer novel targeted delivery system composed of magnetic DNA nanoparticles to be effective as an efficient targeted gene therapy vehicle for tumor therapy. A polysaccharide, dextran, was chosen as the vector of plasmid DNA-encoded NK4 that acts as an HGF-antagonist and anti-angiogenic regulator for inhibitions of tumor growth, invasion, and metastasis. Spermine (Sm) was chemically introduced to the hydroxyl groups of dextran to obtain dextran-Sm. When Fe 2+ solution was added to the mixture of dextran-Sm and a plasmid DNA, homogenous DNA nanoparticles were formed via chemical metal coordination bonding with average size of 230 nm. Characterization of DNA nanoparticles was performed via dynamic light scattering measurement, electrophoretic light scattering measurement, as well as transmission electron microscope. DNA nanoparticles effectively condensed plasmid DNA into nanoparticles and enhanced the stability of DNA, while significantly improved transfection efficiency in vitro and tumor accumulation in vivo. In addition, magnetic DNA nanoparticles exhibited high efficiency in antitumor therapy with regards to tumor growth as well as survival of animals evaluated in the presence of external magnetic field. We conclude that the magnetic properties of these DNA nanoparticles would enhance the tracking of non-viral gene delivery systems when administrated in vivo in a test model. These findings suggest that DNA nanoparticles effectively deliver DNA to tumor and thereby inhibiting tumor growth.

  2. Engineering of magnetic DNA nanoparticles for tumor-targeted therapy

    Energy Technology Data Exchange (ETDEWEB)

    Hosseinkhani, Hossein, E-mail: hosseinkhani@yahoo.com [Graduate Institute of Biomedical Engineering, National Taiwan University of Science and Technology (Taiwan Tech) (China); Chen Yiru [National Yang-Ming University, Department of Biomedical Engineering (China); He Wenjie; Hong Poda [Graduate Institute of Biomedical Engineering, National Taiwan University of Science and Technology (Taiwan Tech) (China); Yu, Dah-Shyong [Nanomedicine Research Center, National Defense Medical Center (China); Domb, Abraham J. [Institute of Drug Research, The Center for Nanoscience and Nanotechnology, School of Pharmacy, Faculty of Medicine, Hebrew University of Jerusalem (Israel)

    2013-01-15

    This study aims to engineer novel targeted delivery system composed of magnetic DNA nanoparticles to be effective as an efficient targeted gene therapy vehicle for tumor therapy. A polysaccharide, dextran, was chosen as the vector of plasmid DNA-encoded NK4 that acts as an HGF-antagonist and anti-angiogenic regulator for inhibitions of tumor growth, invasion, and metastasis. Spermine (Sm) was chemically introduced to the hydroxyl groups of dextran to obtain dextran-Sm. When Fe{sup 2+} solution was added to the mixture of dextran-Sm and a plasmid DNA, homogenous DNA nanoparticles were formed via chemical metal coordination bonding with average size of 230 nm. Characterization of DNA nanoparticles was performed via dynamic light scattering measurement, electrophoretic light scattering measurement, as well as transmission electron microscope. DNA nanoparticles effectively condensed plasmid DNA into nanoparticles and enhanced the stability of DNA, while significantly improved transfection efficiency in vitro and tumor accumulation in vivo. In addition, magnetic DNA nanoparticles exhibited high efficiency in antitumor therapy with regards to tumor growth as well as survival of animals evaluated in the presence of external magnetic field. We conclude that the magnetic properties of these DNA nanoparticles would enhance the tracking of non-viral gene delivery systems when administrated in vivo in a test model. These findings suggest that DNA nanoparticles effectively deliver DNA to tumor and thereby inhibiting tumor growth.

  3. Label-free electrochemical biosensing of small-molecule inhibition on O-GlcNAc glycosylation.

    Science.gov (United States)

    Yang, Yu; Gu, Yuxin; Wan, Bin; Ren, Xiaomin; Guo, Liang-Hong

    2017-09-15

    O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) plays a critical role in modulating protein function in many cellular processes and human diseases such as Alzheimer's disease and type II diabetes, and has emerged as a promising new target. Specific inhibitors of OGT could be valuable tools to probe the biological functions of O-GlcNAcylation, but a lack of robust nonradiometric assay strategies to detect glycosylation, has impeded efforts to identify such compounds. Here we have developed a novel label-free electrochemical biosensor for the detection of peptide O-GlcNAcylation using protease-protection strategy and electrocatalytic oxidation of tyrosine mediated by osmium bipyridine as a signal reporter. There is a large difference in the abilities of proteolysis of the glycosylated and the unglycosylated peptides by protease, thus providing a sensing mechanism for OGT activity. When the O-GlcNAcylation is achieved, the glycosylated peptides cannot be cleaved by proteinase K and result in a high current response on indium tin oxide (ITO) electrode. However, when the O-GlcNAcylation is successfully inhibited using a small molecule, the unglycosylated peptides can be cleaved easily and lead to low current signal. Peptide O-GlcNAcylation reaction was performed in the presence of a well-defined small-molecule OGT inhibitor. The results indicated that the biosensor could be used to screen the OGT inhibitors effectively. Our label-free electrochemical method is a promising candidate for protein glycosylation pathway research in screening small-molecule inhibitors of OGT. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Label-Free Electrochemical Immunoassay for C-Reactive Protein

    Directory of Open Access Journals (Sweden)

    Madasamy Thangamuthu

    2018-03-01

    Full Text Available C-reactive protein (CRP is one of the most expressed proteins in blood during acute phase inflammation, and its minute level increase has also been recognized for the clinical diagnosis of cardio vascular diseases. Unfortunately, the available commercial immunoassays are labour intensive, require large sample volumes, and have practical limitations, such as low stability and high production costs. Hence, we have developed a simple, cost effective, and label-free electrochemical immunoassay for the measurement of CRP in a drop of serum sample using an immunosensor strip made up of a screen printed carbon electrode (SPE modified with anti-CRP functionalized gold nanoparticles (AuNPs. The measurement relies on the decrease of the oxidation current of the redox indicator Fe3+/Fe2+, resulting from the immunoreaction between CRP and anti-CRP. Under optimal conditions, the present immunoassay measures CRP in a linear range from 0.4–200 nM (0.047–23.6 µg mL−1, with a detection limit of 0.15 nM (17 ng mL−1, S/N = 3 and sensitivity of 90.7 nA nM−1, in addition to a good reproducibility and storage stability. The analytical applicability of the presented immunoassay is verified by CRP measurements in human blood serum samples. This work provides the basis for a low-priced, safe, and easy-to-use point-of-care immunosensor assay to measure CRP at clinically relevant concentrations.

  5. Label-free aptamer biosensor for selective detection of thrombin

    Energy Technology Data Exchange (ETDEWEB)

    Na, Weidan; Liu, Xiaotong; Wang, Lei; Su, Xingguang, E-mail: suxg@jlu.edu.cn

    2015-10-29

    We fabricated a novel fluorescence biosensor for the selective detection of thrombin by using bovine serum albumin-capped CdS quantum dots (BSA-CdS QDs). Two kinds of designed DNA (DNA1 and DNA2) could bind to CdS QDs through the electrostatic interaction between DNA and Cd{sup 2+} on the surface of CdS QDs. The obtained DNA/BSA-CdS QDs kept stable in the solution with the fluorescence intensity obviously enhanced. Hairpin structure of DNA1contained two domains, one is the aptamer sequence of thrombin and the other is the complementary sequence of DNA2. When thrombin was added, it would bind to DNA1 and induce the hairpin structure of DNA1 changed into G-quadplex structure. Meanwhile, DNA2 would transfer from the surface of CdS QDs to DNA1 via hybridization, which resulted in the removal of DNA1 and DNA2 from the surface of CdS QDs, and led to the fluorescence intensity of CdS QDs reduced. Thus, the determination of thrombin could be achieved by monitoring the change of the fluorescence intensity of CdS QDs. The present method is simple and fast, and exhibits good selectivity for thrombin over other proteins. We have successfully detected thrombin in human serum samples with satisfactory results. - Highlights: • A novel strategy for the detection of thrombin was established based on BSA-CdS QDs. • DNA could serve as the co-ligands to stabilize CdS QDs and enhance the fluorescence intensity. • Thrombin could change the structure of DNA1 and quench the fluorescence of CdS QDs. • Thrombin in real sample was detected with satisfactory results.

  6. TAL effectors: tools for DNA Targeting

    Czech Academy of Sciences Publication Activity Database

    Jankele, R.; Svoboda, Petr

    2014-01-01

    Roč. 13, č. 5 (2014), s. 409-419 ISSN 2041-2649 R&D Projects: GA ČR(CZ) GBP305/12/G034 Institutional support: RVO:68378050 Keywords : TALE * TALEN * DNA editing Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.670, year: 2014

  7. Targeting DNA Methyltranferases in Urological Tumors

    Directory of Open Access Journals (Sweden)

    Ângela Marques-Magalhães

    2018-04-01

    Full Text Available Urological cancers are a heterogeneous group of malignancies accounting for a considerable proportion of cancer-related morbidity and mortality worldwide. Aberrant epigenetic traits, especially altered DNA methylation patterns constitute a hallmark of these tumors. Nonetheless, these alterations are reversible, and several efforts have been carried out to design and test several epigenetic compounds that might reprogram tumor cell phenotype back to a normal state. Indeed, several DNMT inhibitors are currently under evaluation for therapeutic efficacy in clinical trials. This review highlights the critical role of DNA methylation in urological cancers and summarizes the available data on pre-clinical assays and clinical trials with DNMT inhibitors in bladder, kidney, prostate, and testicular germ cell cancers.

  8. Small molecules, inhibitors of DNA-PK, targeting DNA repair and beyond

    Directory of Open Access Journals (Sweden)

    David eDavidson

    2013-01-01

    Full Text Available Many current chemotherapies function by damaging genomic DNA in rapidly dividing cells ultimately leading to cell death. This therapeutic approach differentially targets cancer cells that generally display rapid cell division compared to normal tissue cells. However, although these treatments are initially effective in arresting tumor growth and reducing tumor burden, resistance and disease progression eventually occur. A major mechanism underlying this resistance is increased levels of cellular DNA repair. Most cells have complex mechanisms in place to repair DNA damage that occurs due to environmental exposures or normal metabolic processes. These systems, initially overwhelmed when faced with chemotherapy induced DNA damage, become more efficient under constant selective pressure and as a result chemotherapies become less effective. Thus, inhibiting DNA repair pathways using target specific small molecule inhibitors may overcome cellular resistance to DNA damaging chemotherapies. Non-homologous end joining (NHEJ a major mechanism for the repair of double strand breaks (DSB in DNA is regulated in part by the serine/threonine kinase, DNA dependent protein kinase (DNA-PK. The DNA-PK holoenzyme acts as a scaffold protein tethering broken DNA ends and recruiting other repair molecules. It also has enzymatic activity that may be involved in DNA damage signaling. Because of its’ central role in repair of DSBs, DNA-PK has been the focus of a number of small molecule studies. In these studies specific DNA-PK inhibitors have shown efficacy in synergizing chemotherapies in vitro. However, compounds currently known to specifically inhibit DNA-PK are limited by poor pharmacokinetics: these compounds have poor solubility and have high metabolic lability in vivo leading to short serum half-lives. Future improvement in DNA-PK inhibition will likely be achieved by designing new molecules based on the recently reported crystallographic structure of DNA

  9. Label-free direct surface-enhanced Raman scattering (SERS) of nucleic acids (Conference Presentation)

    Science.gov (United States)

    Guerrini, Luca; Morla-Folch, Judit; Gisbert-Quilis, Patricia; Xie, Hainan; Alvarez-Puebla, Ramon

    2016-03-01

    Recently, plasmonic-based biosensing has experienced an unprecedented level of attention, with a particular focus on the nucleic acid detection, offering efficient solutions to engineer simple, fast, highly sensitive sensing platforms while overcoming important limitations of PCR and microarray techniques. In the broad field of plasmonics, surface-enhanced Raman scattering (SERS) spectroscopy has arisen as a powerful analytical tool for detection and structural characterization of biomolecules. Today applications of SERS to nucleic acid analysis largely rely on indirect strategies, which have been demonstrated very effective for pure sensing purposes but completely dismiss the exquisite structural information provided by the direct acquisition of the biomolecular vibrational fingerprint. Contrarily, direct label-free SERS of nucleic acid shows an outstanding potential in terms of chemical-specific information which, however, remained largely unexpressed mainly because of the inherent poor spectral reproducibility and/or limited sensitivity. To address these limitations, we developed a fast and affordable high-throughput screening direct SERS method for gaining detailed genomic information on nucleic acids (DNA and RNA) and for the characterization and quantitative recognition of DNA interactions with exogenous agents. The simple strategy relies on the electrostatic adhesion of DNA/RNA onto positively-charged silver colloids that promotes the nanoparticle aggregation into stable clusters yielding intense and reproducible SERS spectra at picogram level (i.e. the analysis can be performed without the necessity of amplification steps thus providing realistic direct information of the nucleic acid in its native state). We anticipate this method to gain a vast impact and set of applications in different fields, including medical diagnostics, genomic screening, drug discovery, forensic science and even molecular electronics.

  10. Label-free imaging of mammalian cell nucleoli by Raman microspectroscopy.

    Science.gov (United States)

    Schulze, H Georg; Konorov, Stanislav O; Piret, James M; Blades, Michael W; Turner, Robin F B

    2013-06-21

    The nucleolus is a prominent subnuclear structure whose major function is the transcription and assembly of ribosome subunits. The size of the nucleolus varies with the cell cycle, proliferation rate and stress. Changes in nucleolar size, number, chemical composition, and shape can be used to characterize malignant cells. We used spontaneous Raman microscopy as a label-free technique to examine nucleolar spatial and chemical features. Raman images of the 1003 cm(-1) phenylalanine band revealed large, well-defined subnuclear protein structures in MFC-7 breast cancer cells. The 783 cm(-1) images showed that nucleic acids were similarly distributed, but varied more in intensity, forming observable high-intensity regions. High subnuclear RNA concentrations were observed within some of these regions as shown by 809 cm(-1) Raman band images. Principal component analyses of sub-images and library spectra validated the subnuclear presence of RNA. They also revealed that an actin-like protein covaried with DNA within the nucleolus, a combination that accounted for 64% or more of the spectral variance. Embryonic stem cells are another rapidly proliferating cell type, but their nucleoli were not as large or well defined. Estimating the size of the larger MCF-7 nucleolus was used to show the utility of Raman microscopy for morphometric analyses. It was concluded that imaging based on Raman microscopy provides a promising new method for the study of nucleolar function and organization, in the evaluation of drug and experimental effects on the nucleolus, and in clinical diagnostics and prognostics.

  11. Label-Free Imaging of Umbilical Cord Tissue Morphology and Explant-Derived Cells

    Directory of Open Access Journals (Sweden)

    Raf Donders

    2016-01-01

    Full Text Available In situ detection of MSCs remains difficult and warrants additional methods to aid with their characterization in vivo. Two-photon confocal laser scanning microscopy (TPM and second harmonic generation (SHG could fill this gap. Both techniques enable the detection of cells and extracellular structures, based on intrinsic properties of the specific tissue and intracellular molecules under optical irradiation. TPM imaging and SHG imaging have been used for label-free monitoring of stem cells differentiation, assessment of their behavior in biocompatible scaffolds, and even cell tracking in vivo. In this study, we show that TPM and SHG can accurately depict the umbilical cord architecture and visualize individual cells both in situ and during culture initiation, without the use of exogenously applied labels. In combination with nuclear DNA staining, we observed a variance in fluorescent intensity in the vessel walls. In addition, antibody staining showed differences in Oct4, αSMA, vimentin, and ALDH1A1 expression in situ, indicating functional differences among the umbilical cord cell populations. In future research, marker-free imaging can be of great added value to the current antigen-based staining methods for describing tissue structures and for the identification of progenitor cells in their tissue of origin.

  12. A systematic evaluation of normalization methods in quantitative label-free proteomics.

    Science.gov (United States)

    Välikangas, Tommi; Suomi, Tomi; Elo, Laura L

    2018-01-01

    To date, mass spectrometry (MS) data remain inherently biased as a result of reasons ranging from sample handling to differences caused by the instrumentation. Normalization is the process that aims to account for the bias and make samples more comparable. The selection of a proper normalization method is a pivotal task for the reliability of the downstream analysis and results. Many normalization methods commonly used in proteomics have been adapted from the DNA microarray techniques. Previous studies comparing normalization methods in proteomics have focused mainly on intragroup variation. In this study, several popular and widely used normalization methods representing different strategies in normalization are evaluated using three spike-in and one experimental mouse label-free proteomic data sets. The normalization methods are evaluated in terms of their ability to reduce variation between technical replicates, their effect on differential expression analysis and their effect on the estimation of logarithmic fold changes. Additionally, we examined whether normalizing the whole data globally or in segments for the differential expression analysis has an effect on the performance of the normalization methods. We found that variance stabilization normalization (Vsn) reduced variation the most between technical replicates in all examined data sets. Vsn also performed consistently well in the differential expression analysis. Linear regression normalization and local regression normalization performed also systematically well. Finally, we discuss the choice of a normalization method and some qualities of a suitable normalization method in the light of the results of our evaluation. © The Author 2016. Published by Oxford University Press.

  13. Label-free imaging of gold nanoparticles in single live cells by photoacoustic microscopy

    Science.gov (United States)

    Tian, Chao; Qian, Wei; Shao, Xia; Xie, Zhixing; Cheng, Xu; Liu, Shengchun; Cheng, Qian; Liu, Bing; Wang, Xueding

    2016-03-01

    Gold nanoparticles (AuNPs) have been extensively explored as a model nanostructure in nanomedicine and have been widely used to provide advanced biomedical research tools in diagnostic imaging and therapy. Due to the necessity of targeting AuNPs to individual cells, evaluation and visualization of AuNPs in the cellular level is critical to fully understand their interaction with cellular environment. Currently imaging technologies, such as fluorescence microscopy and transmission electron microscopy all have advantages and disadvantages. In this paper, we synthesized AuNPs by femtosecond pulsed laser ablation, modified their surface chemistry through sequential bioconjugation, and targeted the functionalized AuNPs with individual cancer cells. Based on their high optical absorption contrast, we developed a novel, label-free imaging method to evaluate and visualize intracellular AuNPs using photoacoustic microscopy (PAM). Preliminary study shows that the PAM imaging technique is capable of imaging cellular uptake of AuNPs in vivo at single-cell resolution, which provide an important tool for the study of AuNPs in nanomedicine.

  14. Sensitive and label-free detection of miRNA-145 by triplex formation.

    Science.gov (United States)

    Aviñó, Anna; Huertas, César S; Lechuga, Laura M; Eritja, Ramon

    2016-01-01

    The development of new strategies for detecting microRNAs (miRNAs) has become a crucial step in the diagnostic field. miRNA profiles depend greatly on the sample and the analytical platform employed, leading sometimes to contradictory results. In this work, we study the use of modified parallel tail-clamps to detect a miRNA sequence involved in tumor suppression by triplex formation. Thermal denaturing curves and circular dichroism (CD) measurements have been performed to confirm that parallel clamps carrying 8-aminoguanine form the most stable triplex structures with their target miRNA. The modified tail-clamps have been tested as bioreceptors in a surface plasmon resonance (SPR) biosensor for the detection of miRNA-145. The detection limit was improved 2.4 times demonstrating that a stable triplex structure is formed between target miRNA and 8-aminoguanine tail-clamp bioreceptor. This new approach is an essential step toward the label-free and reliable detection of miRNA signatures for diagnostic purposes.

  15. "Molecular beacon"-hosted thioflavin T: Applications for label-free fluorescent detection of iodide and logic operations.

    Science.gov (United States)

    Li, Yan-Yun; Jiang, Xiao-Qin; Lu, Ling-Fei; Zhang, Min; Shi, Guoyue

    2016-04-01

    In this work, we presented a simple, label-free and rapid-responsive fluorescence assay for iodide (I(-)) detection based on "molecular beacon (MB)"-hosted thioflavin T (ThT), achieving a limit of detection as low as 158 nM. The proposed method exhibited very good selectivity to I(-) ions over other anions interference due to the strong binding force between I(-) ions with Hg(2+). Upon the addition of I(-) ions, it would capture Hg(2+) from a T-Hg(2+)-T complex belonging to the MB-like DNA hairpin structure, which eventually quenched the initial fluorescence as output. In addition, it was successfully applied for operation of an integrated DNA logic gate system and to the determination of I(-) in real samples such as human urine. Copyright © 2016 Elsevier B.V. All rights reserved.

  16. DNA-PK dependent targeting of DNA-ends to a protein complex assembled on matrix attachment region DNA sequences

    International Nuclear Information System (INIS)

    Mauldin, S.K.; Getts, R.C.; Perez, M.L.; DiRienzo, S.; Stamato, T.D.

    2003-01-01

    Full text: We find that nuclear protein extracts from mammalian cells contain an activity that allows DNA ends to associate with circular pUC18 plasmid DNA. This activity requires the catalytic subunit of DNA-PK (DNA-PKcs) and Ku since it was not observed in mutants lacking Ku or DNA-PKcs but was observed when purified Ku/DNA-PKcs was added to these mutant extracts. Competition experiments between pUC18 and pUC18 plasmids containing various nuclear matrix attachment region (MAR) sequences suggest that DNA ends preferentially associate with plasmids containing MAR DNA sequences. At a 1:5 mass ratio of MAR to pUC18, approximately equal amounts of DNA end binding to the two plasmids were observed, while at a 1:1 ratio no pUC18 end-binding was observed. Calculation of relative binding activities indicates that DNA-end binding activities to MAR sequences was 7 to 21 fold higher than pUC18. Western analysis of proteins bound to pUC18 and MAR plasmids indicates that XRCC4, DNA ligase IV, scaffold attachment factor A, topoisomerase II, and poly(ADP-ribose) polymerase preferentially associate with the MAR plasmid in the absence or presence of DNA ends. In contrast, Ku and DNA-PKcs were found on the MAR plasmid only in the presence of DNA ends. After electroporation of a 32P-labeled DNA probe into human cells and cell fractionation, 87% of the total intercellular radioactivity remained in nuclei after a 0.5M NaCl extraction suggesting the probe was strongly bound in the nucleus. The above observations raise the possibility that DNA-PK targets DNA-ends to a repair and/or DNA damage signaling complex which is assembled on MAR sites in the nucleus

  17. Cell and brain tissue imaging of the flavonoid fisetin using label-free two-photon microscopy.

    Science.gov (United States)

    Krasieva, Tatiana B; Ehren, Jennifer; O'Sullivan, Thomas; Tromberg, Bruce J; Maher, Pamela

    2015-10-01

    Over the last few years, we have identified an orally active, novel neuroprotective and cognition-enhancing molecule, the flavonoid fisetin. Fisetin not only has direct antioxidant activity but it can also increase the intracellular levels of glutathione, the major intracellular antioxidant. Fisetin can also activate key neurotrophic factor signaling pathways. In addition, it has anti-inflammatory activity against microglia and astrocytes and inhibits the activity of lipoxygenases, thereby reducing the production of pro-inflammatory eicosanoids and their by-products. However, key questions about its targets and brain penetration remain. In this study, we used label-free two-photon microscopy of intrinsic fisetin fluorescence to examine the localization of fisetin in living nerve cells and the brains of living mice. In cells, fisetin but not structurally related flavonols with different numbers of hydroxyl groups, localized to the nucleoli suggesting that key targets of fisetin may reside in this organelle. In the mouse brain, following intraperitoneal injection and oral administration, fisetin rapidly distributed to the blood vessels of the brain followed by a slower dispersion into the brain parenchyma. Thus, these results provide further support for the effects of fisetin on brain function. In addition, they suggest that label-free two-photon microscopy may prove useful for studying the intracellular and tissue distribution of other intrinsically-fluorescent flavonoids. Copyright © 2015 Elsevier Ltd. All rights reserved.

  18. Label-Free Detection of Bacillus anthracis Spore Uptake in Macrophage Cells Using Analytical Optical Force Measurements.

    Science.gov (United States)

    Hebert, Colin G; Hart, Sean; Leski, Tomasz A; Terray, Alex; Lu, Qin

    2017-10-03

    Understanding the interaction between macrophage cells and Bacillus anthracis spores is of significant importance with respect to both anthrax disease progression, spore detection for biodefense, as well as understanding cell clearance in general. While most detection systems rely on specific molecules, such as nucleic acids or proteins and fluorescent labels to identify the target(s) of interest, label-free methods probe changes in intrinsic properties, such as size, refractive index, and morphology, for correlation with a particular biological event. Optical chromatography is a label free technique that uses the balance between optical and fluidic drag forces within a microfluidic channel to determine the optical force on cells or particles. Here we show an increase in the optical force experienced by RAW264.7 macrophage cells upon the uptake of both microparticles and B. anthracis Sterne 34F2 spores. In the case of spores, the exposure was detected in as little as 1 h without the use of antibodies or fluorescent labels of any kind. An increase in the optical force was also seen in macrophage cells treated with cytochalasin D, both with and without a subsequent exposure to spores, indicating that a portion of the increase in the optical force arises independent of phagocytosis. These results demonstrate the capability of optical chromatography to detect subtle biological differences in a rapid and sensitive manner and suggest future potential in a range of applications, including the detection of biological threat agents for biodefense and pathogens for the prevention of sepsis and other diseases.

  19. DNA repair systems as targets of cadmium toxicity

    International Nuclear Information System (INIS)

    Giaginis, Constantinos; Gatzidou, Elisavet; Theocharis, Stamatios

    2006-01-01

    Cadmium (Cd) is a heavy metal and a potent carcinogen implicated in tumor development through occupational and environmental exposure. Recent evidence suggests that proteins participating in the DNA repair systems, especially in excision and mismatch repair, are sensitive targets of Cd toxicity. Cd by interfering and inhibiting these DNA repair processes might contribute to increased risk for tumor formation in humans. In the present review, the information available on the interference of Cd with DNA repair systems and their inhibition is summarized. These actions could possibly explain the indirect contribution of Cd to mutagenic effects and/or carcinogenicity

  20. A miniaturized optoelectronic system for rapid quantitative label-free detection of harmful species in food

    Science.gov (United States)

    Raptis, Ioannis; Misiakos, Konstantinos; Makarona, Eleni; Salapatas, Alexandros; Petrou, Panagiota; Kakabakos, Sotirios; Botsialas, Athanasios; Jobst, Gerhard; Haasnoot, Willem; Fernandez-Alba, Amadeo; Lees, Michelle; Valamontes, Evangelos

    2016-03-01

    Optical biosensors have emerged in the past decade as the most promising candidates for portable, highly-sensitive bioanalytical systems that can be employed for in-situ measurements. In this work, a miniaturized optoelectronic system for rapid, quantitative, label-free detection of harmful species in food is presented. The proposed system has four distinctive features that can render to a powerful tool for the next generation of Point-of-Need applications, namely it accommodates the light sources and ten interferometric biosensors on a single silicon chip of a less-than-40mm2 footprint, each sensor can be individually functionalized for a specific target analyte, the encapsulation can be performed at the wafer-scale, and finally it exploits a new operation principle, Broad-band Mach-Zehnder Interferometry to ameliorate its analytical capabilities. Multi-analyte evaluation schemes for the simultaneous detection of harmful contaminants, such as mycotoxins, allergens and pesticides, proved that the proposed system is capable of detecting within short time these substances at concentrations below the limits imposed by regulatory authorities, rendering it to a novel tool for the near-future food safety applications.

  1. Label-free signal-on aptasensor for sensitive electrochemical detection of arsenite.

    Science.gov (United States)

    Cui, Lin; Wu, Jie; Ju, Huangxian

    2016-05-15

    A signal-on aptasensor was fabricated for highly sensitive and selective electrochemical detection of arsenite with a label-free Ars-3 aptamer self-assembled on a screen-printed carbon electrode (SPCE) via Au-S bond. The Ars-3 aptamer could adsorb cationic polydiallyldimethylammonium (PDDA) via electrostatic interaction to repel other cationic species. In the presence of arsenite, the change of Ars-3 conformation due to the formation of Ars-3/arsenite complex led to less adsorption of PDDA, and the complex could adsorb more positively charged [Ru(NH3)6](3+) as an electrochemically active indicator on the aptasensor surface, which produced a sensitive "turn-on" response. The target-induced structure switching could be used for sensitive detection of arsenite with a linear range from 0.2 nM to 100 nM and a detection limit down to 0.15 nM. Benefiting from Ars-3 aptamer, the proposed system exhibited excellent specificity against other heavy metal ions. The SPCE-based aptasensor exhibited the advantages of low cost and simple fabrication, providing potential application of arsenite detection in environment. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. Theoretical analysis of bimetallic nanorod dimer biosensors for label-free molecule detection

    Science.gov (United States)

    Das, Avijit; Talukder, Muhammad Anisuzzaman

    2018-02-01

    In this work, we theoretically analyze a gold (Au) core within silver (Ag) shell (Au@Ag) nanorod dimer biosensor for label-free molecule detection. The incident light on an Au@Ag nanorod strongly couples to localized surface plasmon modes, especially around the tip region. The field enhancement around the tip of a nanorod or between the tips of two longitudinally aligned nanorods as in a dimer can be exploited for sensitive detection of biomolecules. We derive analytical expressions for the interactions of an Au@Ag nanorod dimer with the incident light. We also study the detail dynamics of an Au@Ag nanorod dimer with the incident light computationally using finite difference time domain (FDTD) technique when core-shell ratio, relative position of the nanorods, and angle of incidence of light change. We find that the results obtained using the developed analytical model match well with that obtained using FDTD simulations. Additionally, we investigate the sensitivity of the Au@Ag nanorod dimer, i.e., shift in the resonance wavelength, when a target biomolecule such as lysozyme (Lys), human serum albumin (HSA), anti-biotin (Abn), human catalase (CAT), and human fibrinogen (Fb) protein molecules are attached to the tips of the nanorods.

  3. Fully integrated graphene electronic biosensor for label-free detection of lead (II) ion based on G-quadruplex structure-switching.

    Science.gov (United States)

    Li, Yijun; Wang, Cheng; Zhu, Yibo; Zhou, Xiaohong; Xiang, Yu; He, Miao; Zeng, Siyu

    2017-03-15

    This work presents a fully integrated graphene field-effect transistor (GFET) biosensor for the label-free detection of lead ions (Pb 2+ ) in aqueous-media, which first implements the G-quadruplex structure-switching biosensing principle in graphene nanoelectronics. We experimentally illustrate the biomolecular interplay that G-rich DNA single-strands with one-end confined on graphene surface can specifically interact with Pb 2+ ions and switch into G-quadruplex structures. Since the structure-switching of electrically charged DNA strands can disrupt the charge distribution in the vicinity of graphene surface, the carrier equilibrium in graphene sheet might be altered, and manifested by the conductivity variation of GFET. The experimental data and theoretical analysis show that our devices are capable of the label-free and specific quantification of Pb 2+ with a detection limit down to 163.7ng/L. These results first verify the signaling principle competency of G-quadruplex structure-switching in graphene electronic biosensors. Combining with the advantages of the compact device structure and convenient electrical signal, a label-free GFET biosensor for Pb 2+ monitoring is enabled with promising application potential. Copyright © 2016 Elsevier B.V. All rights reserved.

  4. Real-time label-free quantitative fluorescence microscopy-based detection of ATP using a tunable fluorescent nano-aptasensor platform.

    Science.gov (United States)

    Shrivastava, Sajal; Sohn, Il-Yung; Son, Young-Min; Lee, Won-Il; Lee, Nae-Eung

    2015-12-14

    Although real-time label-free fluorescent aptasensors based on nanomaterials are increasingly recognized as a useful strategy for the detection of target biomolecules with high fidelity, the lack of an imaging-based quantitative measurement platform limits their implementation with biological samples. Here we introduce an ensemble strategy for a real-time label-free fluorescent graphene (Gr) aptasensor platform. This platform employs aptamer length-dependent tunability, thus enabling the reagentless quantitative detection of biomolecules through computational processing coupled with real-time fluorescence imaging data. We demonstrate that this strategy effectively delivers dose-dependent quantitative readouts of adenosine triphosphate (ATP) concentration on chemical vapor deposited (CVD) Gr and reduced graphene oxide (rGO) surfaces, thereby providing cytotoxicity assessment. Compared with conventional fluorescence spectrometry methods, our highly efficient, universally applicable, and rational approach will facilitate broader implementation of imaging-based biosensing platforms for the quantitative evaluation of a range of target molecules.

  5. Real-time label-free quantitative fluorescence microscopy-based detection of ATP using a tunable fluorescent nano-aptasensor platform

    Science.gov (United States)

    Shrivastava, Sajal; Sohn, Il-Yung; Son, Young-Min; Lee, Won-Il; Lee, Nae-Eung

    2015-11-01

    Although real-time label-free fluorescent aptasensors based on nanomaterials are increasingly recognized as a useful strategy for the detection of target biomolecules with high fidelity, the lack of an imaging-based quantitative measurement platform limits their implementation with biological samples. Here we introduce an ensemble strategy for a real-time label-free fluorescent graphene (Gr) aptasensor platform. This platform employs aptamer length-dependent tunability, thus enabling the reagentless quantitative detection of biomolecules through computational processing coupled with real-time fluorescence imaging data. We demonstrate that this strategy effectively delivers dose-dependent quantitative readouts of adenosine triphosphate (ATP) concentration on chemical vapor deposited (CVD) Gr and reduced graphene oxide (rGO) surfaces, thereby providing cytotoxicity assessment. Compared with conventional fluorescence spectrometry methods, our highly efficient, universally applicable, and rational approach will facilitate broader implementation of imaging-based biosensing platforms for the quantitative evaluation of a range of target molecules.Although real-time label-free fluorescent aptasensors based on nanomaterials are increasingly recognized as a useful strategy for the detection of target biomolecules with high fidelity, the lack of an imaging-based quantitative measurement platform limits their implementation with biological samples. Here we introduce an ensemble strategy for a real-time label-free fluorescent graphene (Gr) aptasensor platform. This platform employs aptamer length-dependent tunability, thus enabling the reagentless quantitative detection of biomolecules through computational processing coupled with real-time fluorescence imaging data. We demonstrate that this strategy effectively delivers dose-dependent quantitative readouts of adenosine triphosphate (ATP) concentration on chemical vapor deposited (CVD) Gr and reduced graphene oxide (r

  6. Using label-free screening technology to improve efficiency in drug discovery.

    Science.gov (United States)

    Halai, Reena; Cooper, Matthew A

    2012-02-01

    Screening assays have traditionally utilized reporter labels to quantify biological responses relevant to the disease state of interest. However, there are limitations associated with the use of labels that may be overcome with temporal measurements possible with label-free. This review comprises general and system-specific information from literature searches using PubMed, published books and the authors' personal experience. This review highlights the label-free approaches in the context of various applications. The authors also note technical issues relevant to the development of label-free assays and their application to HTS. The limitations associated with the use of transfected cell lines and the use of label-based assays are gradually being realized. As such, greater emphasis is being placed on label-free biophysical techniques using native cell lines. The introduction of 96- and 384-well plate label-free systems is helping to broker a wider acceptance of these approaches in high-throughput screening. However, potential users of the technologies remain skeptical, primarily because the physical basis of the signals generated, and their contextual relevance to cell biology and signal transduction, has not been fully elucidated. Until this is done, these new technology platforms are more likely to complement, rather than replace, traditional screening platforms.

  7. Extracting histones for the specific purpose of label-free MS.

    Science.gov (United States)

    Govaert, Elisabeth; Van Steendam, Katleen; Scheerlinck, Ellen; Vossaert, Liesbeth; Meert, Paulien; Stella, Martina; Willems, Sander; De Clerck, Laura; Dhaenens, Maarten; Deforce, Dieter

    2016-12-01

    Extracting histones from cells is the first step in studies that aim to characterize histones and their post-translational modifications (hPTMs) with MS. In the last decade, label-free quantification is more frequently being used for MS-based histone characterization. However, many histone extraction protocols were not specifically designed for label-free MS. While label-free quantification has its advantages, it is also very susceptible to technical variation. Here, we adjust an established histone extraction protocol according to general label-free MS guidelines with a specific focus on minimizing sample handling. These protocols are first evaluated using SDS-PAGE. Hereafter, a selection of extraction protocols was used in a complete histone workflow for label-free MS. All protocols display nearly identical relative quantification of hPTMs. We thus show that, depending on the cell type under investigation and at the cost of some additional contaminating proteins, minimizing sample handling can be done during histone isolation. This allows analyzing bigger sample batches, leads to reduced technical variation and minimizes the chance of in vitro alterations to the hPTM snapshot. Overall, these results allow researchers to determine the best protocol depending on the resources and goal of their specific study. Data are available via ProteomeXchange with identifier PXD002885. © 2016 The Authors. Proteomics Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. A label-free photoelectrochemical cocaine aptasensor based on an electropolymerized ruthenium-intercalator complex

    International Nuclear Information System (INIS)

    Haddache, Fatima; Le Goff, Alan; Spinelli, Nicolas; Gairola, Priyanka; Gorgy, Karine; Gondran, Chantal; Defrancq, Eric; Cosnier, Serge

    2016-01-01

    Highlights: • Electrodes were modified by an electrogenerated Ru(II) complex which demonstrates photosensitive properties and intercalating properties towards the stem-loop base pairing domain of cocaine aptamers. • Cocaine aptamers were immobilized as mono-and double-fragment which showed different behaviour towards photocurrent generation. • The binding of aptamer could be followed by photelectrochemistry and modelized using a Langmuir-Freundlich isotherm. • Using the double-fragment aptamer, a label-free photoelectrochemical aptasensor was designed, exhibiting a LOD of 10 nmol L −1 and linear range of 1 10 −8 –5 10 −4 mol L −1 . - Abstract: A photoelectrode was designed by electrodeposition of a pyrrole monomer modified with a polypyridyl Ru(II) complex bearing benzo[i]dipyrido-[3,2-a:2′.3′-c]phenazine (dppn) ligand. Owing to the intercalating properties of these immobilized complexes towards DNA double helix, cocaine aptamer was immobilized on the modified electrodes thanks to its stem-loop configuration in order to design a photoelectrochemical cocaine aptasensor. Especially using a double-fragment aptamer strategy, the binding of cocaine and the formation of the aptamer/cocaine complex was successfully observed and modeled by a Langmuir-Freundlich isotherm, giving access to an apparent dissociation constant K d of 3.8 mmol L −1 . The photoelectrochemical aptasensor exhibits a LOD of 10 nmol L −1 and linear range of 1 10 −8 –5 10 −4 mol L −1 .

  9. Label-free fluorimetric detection of CEA using carbon dots derived from tomato juice.

    Science.gov (United States)

    Miao, Hong; Wang, Lan; Zhuo, Yan; Zhou, Zinan; Yang, Xiaoming

    2016-12-15

    A facile-green strategy to synthesize carbon dots (CDs) with a quantum yield (QY) of nearly 13.9% has been built up, while tomato juice served as the carbon source. Interestingly, not only the precursor of CDs and the whole synthesis procedure were environmental-friendly, but this type of CDs also exhibited multiple advantages including high fluorescent QY, excellent photostability, non-toxicity and satisfactory stability. Significantly, a label-free sensitive assay for detecting carcinoembryonic antigen (CEA) in a continuous and recyclable way has been proposed on the basis of adsorption and desorption of aptamers by the surface of CDs through a competitive mechanism. To be specific, the richness of carboxyl groups of the CDs enabled strong adsorption of ssDNA to the surface of CDs through π-π stacking interactions, resulting in the effective fluorescence quenching by forming CDs-aptamer complexes. The stronger binding affinity between CEA and CEA-aptamer than the π-π stacking interactions has been taken advantage to achieve immediate recovery of the fluorescence of CDs once CEA was introduced. Thereby, quantitative evaluation of CEA concentration in a broad range from 1ngmL(-1) to 0.5ngmL(-1) with the detection limit of 0.3ngmL(-1) was realized in this way. This strategy can be applied in a recyclable way, broadening the sensing application of CDs with biocompatibility. Besides, the CDs were used for cell imaging, potentiating them towards diverse purposes. Copyright © 2016 Elsevier B.V. All rights reserved.

  10. Highly multiplexed targeted DNA sequencing from single nuclei.

    Science.gov (United States)

    Leung, Marco L; Wang, Yong; Kim, Charissa; Gao, Ruli; Jiang, Jerry; Sei, Emi; Navin, Nicholas E

    2016-02-01

    Single-cell DNA sequencing methods are challenged by poor physical coverage, high technical error rates and low throughput. To address these issues, we developed a single-cell DNA sequencing protocol that combines flow-sorting of single nuclei, time-limited multiple-displacement amplification (MDA), low-input library preparation, DNA barcoding, targeted capture and next-generation sequencing (NGS). This approach represents a major improvement over our previous single nucleus sequencing (SNS) Nature Protocols paper in terms of generating higher-coverage data (>90%), thereby enabling the detection of genome-wide variants in single mammalian cells at base-pair resolution. Furthermore, by pooling 48-96 single-cell libraries together for targeted capture, this approach can be used to sequence many single-cell libraries in parallel in a single reaction. This protocol greatly reduces the cost of single-cell DNA sequencing, and it can be completed in 5-6 d by advanced users. This single-cell DNA sequencing protocol has broad applications for studying rare cells and complex populations in diverse fields of biological research and medicine.

  11. Real-time monitoring of mycobacterium genomic DNA with target-primed rolling circle amplification by a Au nanoparticle-embedded SPR biosensor.

    Science.gov (United States)

    Xiang, Yang; Zhu, Xiaoyan; Huang, Qing; Zheng, Junsong; Fu, Weiling

    2015-04-15

    In this study, we developed a surface plasmon resonance (SPR) DNA biosensor array based on target-primed rolling circle amplification (RCA) for isothermal and rapid detection of two pathogenic mycobacteria, Mycobacterium tuberculosis complex (MTBC) and Mycobacterium avium complex (MAC).The species-specific padlock probe (PLP) was designed to target the sequence in 16S-23S rRNA gene internal transcribed spacer (ITS). After ligation, the circularized PLP could be primed by the target sequence to initial RCA. The RCA performed simultaneously with the cleavage reaction to produce small fragments of single strand DNA which immediately hybridized with the probe immobilized on the sensor chip without denaturation. This process caused SPR angle changes on the chip surface, which made the detection for analysis from the solution achievable, and dynamic real-time RCA monitoring of mycobacterium possible. Besides, Au nanoparticles (AuNPs) were directly assembled onto the surface of the sensor chip via hexanedithiol (HDT) for the enhancement of sensitivity as a label-free detection system. Experimental results show that the signal enhancement by the target-primed RCA together with AuNPs-embedded surface caused at least10-fold increased sensitivity as compared with conventional RCA on bare SPR chip method. Within 40min amplification duration as low as 20amol of synthetic targets and 10(4)CFUmL(-1) of genomic DNA from clinical samples can be detected. The proposed method not only provides a simple design idea for liquid-phase amplification monitoring, but also apply it in clinical pathogen detection, which holds great promise in ultrasensitive bioassay in the future. Copyright © 2014. Published by Elsevier B.V.

  12. Targeting telomerase and DNA repair in human cancers

    International Nuclear Information System (INIS)

    Prakash Hande, M.

    2014-01-01

    Telomerase reactivation is essential for telomere maintenance in human cancer cells ensuring indefinite proliferation. Targeting telomere homeostasis has become one of the promising strategies in the therapeutic management of tumours. One major potential drawback, however, is the time lag between telomerase inhibition and critically shortened telomeres triggering cell death, allowing cancer cells to acquire drug resistance. Numerous studies over the last decade have highlighted the role of DNA repair proteins such as Poly (ADP-Ribose) Polymerase-1 (PARP-1), and DNA-dependent protein kinase (DNA-PKcs) in the maintenance of telomere homoeostasis. Dysfunctional telomeres, resulting from the loss of telomeric DNA repeats or the loss of function of telomere-associated proteins trigger DNA damage responses similar to that observed for double strand breaks. We have been working on unravelling such synthetic lethality in cancer cells and this talk would be on one such recently concluded study that demonstrates that inhibition of DNA repair pathways, i.e., NHEJ pathway and that of telomerase could be an alternative strategy to enhance anti-tumour effects and circumvent the possibility of drug resistance. (author)

  13. iTRAQ-Based and Label-Free Proteomics Approaches for Studies of Human Adenovirus Infections

    OpenAIRE

    Trinh, Hung V.; Grossmann, Jonas; Gehrig, Peter; Roschitzki, Bernd; Schlapbach, Ralph; Greber, Urs F.; Hemmi, Silvio

    2013-01-01

    Both isobaric tags for relative and absolute quantitation (iTRAQ) and label-free methods are widely used for quantitative proteomics. Here, we provide a detailed evaluation of these proteomics approaches based on large datasets from biological samples. iTRAQ-label-based and label-free quantitations were compared using protein lysate samples from noninfected human lung epithelial A549 cells and from cells infected for 24 h with human adenovirus type 3 or type 5. Either iTRAQ-label-based or lab...

  14. Small-Molecule Inhibitors Targeting DNA Repair and DNA Repair Deficiency in Research and Cancer Therapy.

    Science.gov (United States)

    Hengel, Sarah R; Spies, M Ashley; Spies, Maria

    2017-09-21

    To maintain stable genomes and to avoid cancer and aging, cells need to repair a multitude of deleterious DNA lesions, which arise constantly in every cell. Processes that support genome integrity in normal cells, however, allow cancer cells to develop resistance to radiation and DNA-damaging chemotherapeutics. Chemical inhibition of the key DNA repair proteins and pharmacologically induced synthetic lethality have become instrumental in both dissecting the complex DNA repair networks and as promising anticancer agents. The difficulty in capitalizing on synthetically lethal interactions in cancer cells is that many potential targets do not possess well-defined small-molecule binding determinates. In this review, we discuss several successful campaigns to identify and leverage small-molecule inhibitors of the DNA repair proteins, from PARP1, a paradigm case for clinically successful small-molecule inhibitors, to coveted new targets, such as RAD51 recombinase, RAD52 DNA repair protein, MRE11 nuclease, and WRN DNA helicase. Copyright © 2017 Elsevier Ltd. All rights reserved.

  15. Viral interference with DNA repair by targeting of the single-stranded DNA binding protein RPA.

    Science.gov (United States)

    Banerjee, Pubali; DeJesus, Rowena; Gjoerup, Ole; Schaffhausen, Brian S

    2013-10-01

    Correct repair of damaged DNA is critical for genomic integrity. Deficiencies in DNA repair are linked with human cancer. Here we report a novel mechanism by which a virus manipulates DNA damage responses. Infection with murine polyomavirus sensitizes cells to DNA damage by UV and etoposide. Polyomavirus large T antigen (LT) alone is sufficient to sensitize cells 100 fold to UV and other kinds of DNA damage. This results in activated stress responses and apoptosis. Genetic analysis shows that LT sensitizes via the binding of its origin-binding domain (OBD) to the single-stranded DNA binding protein replication protein A (RPA). Overexpression of RPA protects cells expressing OBD from damage, and knockdown of RPA mimics the LT phenotype. LT prevents recruitment of RPA to nuclear foci after DNA damage. This leads to failure to recruit repair proteins such as Rad51 or Rad9, explaining why LT prevents repair of double strand DNA breaks by homologous recombination. A targeted intervention directed at RPA based on this viral mechanism could be useful in circumventing the resistance of cancer cells to therapy.

  16. Sequence heterogeneity accelerates protein search for targets on DNA

    International Nuclear Information System (INIS)

    Shvets, Alexey A.; Kolomeisky, Anatoly B.

    2015-01-01

    The process of protein search for specific binding sites on DNA is fundamentally important since it marks the beginning of all major biological processes. We present a theoretical investigation that probes the role of DNA sequence symmetry, heterogeneity, and chemical composition in the protein search dynamics. Using a discrete-state stochastic approach with a first-passage events analysis, which takes into account the most relevant physical-chemical processes, a full analytical description of the search dynamics is obtained. It is found that, contrary to existing views, the protein search is generally faster on DNA with more heterogeneous sequences. In addition, the search dynamics might be affected by the chemical composition near the target site. The physical origins of these phenomena are discussed. Our results suggest that biological processes might be effectively regulated by modifying chemical composition, symmetry, and heterogeneity of a genome

  17. Sequence heterogeneity accelerates protein search for targets on DNA

    Energy Technology Data Exchange (ETDEWEB)

    Shvets, Alexey A.; Kolomeisky, Anatoly B., E-mail: tolya@rice.edu [Department of Chemistry and Center for Theoretical Biological Physics, Rice University, Houston, Texas 77005 (United States)

    2015-12-28

    The process of protein search for specific binding sites on DNA is fundamentally important since it marks the beginning of all major biological processes. We present a theoretical investigation that probes the role of DNA sequence symmetry, heterogeneity, and chemical composition in the protein search dynamics. Using a discrete-state stochastic approach with a first-passage events analysis, which takes into account the most relevant physical-chemical processes, a full analytical description of the search dynamics is obtained. It is found that, contrary to existing views, the protein search is generally faster on DNA with more heterogeneous sequences. In addition, the search dynamics might be affected by the chemical composition near the target site. The physical origins of these phenomena are discussed. Our results suggest that biological processes might be effectively regulated by modifying chemical composition, symmetry, and heterogeneity of a genome.

  18. Mannosylated biodegradable polyethyleneimine for targeted DNA delivery to dendritic cells

    Directory of Open Access Journals (Sweden)

    Sun X

    2012-06-01

    Full Text Available Xun Sun, Simu Chen, Jianfeng Han, Zhirong ZhangKey Laboratory of Drug Targeting and Drug Delivery System, Ministry of Education, West China School of Pharmacy, Sichuan University, Chengdu, People’s Republic of ChinaBackground: To establish a potential gene-delivery system with the ability to deliver plasmid DNA to dendritic cells (DCs more efficiently and specifically, we designed and synthesized a low-molecular-weight polyethyleneimine and triethyleneglycol polymer (PEI–TEG and a series of its mannosylated derivatives.Methods: PEI–TEG was synthesized from PEI2000 and PEI600 with TEG as the cross-linker. PEI–TEG was then linked to mannose via a phenylisothiocyanate bridge to obtain man-PEI–TEG conjugates. The DNA conveyance abilities of PEI–TEG, man-PEI–TEG, as well as control PEI25k were evaluated by measuring their zeta potential, particle size, and DNA-binding abilities. The in vitro cytotoxicity, cell uptake, and transfection efficiency of these PEI/DNA complexes were examined on the DC2.4 cell line. Finally, a maturation experiment evaluated the effect of costimulatory molecules CD40, CD80, and CD86 on murine bone marrow-derived DCs (BMDCs using flow cytometry.Results: PEI–TEG and man-PEI–TEG were successfully synthesized and were shown to retain the excellent properties of PEI25k for condensing DNA. Compared with PEI–TEG as well as PEI25k, the man-PEI–TEG had less cytotoxicity and performed better in both cellular uptake and transfection assays in vitro. The results of the maturation experiment showed that all the PEI/DNA complexes induced an adequate upregulation of surface markers for DC maturation.Conclusion: These results demonstrated that man-PEI–TEG can be employed as a DC-targeting gene-delivery system.Keywords: dendritic cells, DCs, mannose, polyethyleneimine, PEI, gene delivery

  19. Label-free biosensing with functionalized nanopipette probes.

    Science.gov (United States)

    Umehara, Senkei; Karhanek, Miloslav; Davis, Ronald W; Pourmand, Nader

    2009-03-24

    Nanopipette technology can uniquely identify biomolecules such as proteins based on differences in size, shape, and electrical charge. These differences are determined by the detection of changes in ionic current as the proteins interact with the nanopipette tip coated with probe molecules. Here we show that electrostatic, biotin-streptavidin, and antibody-antigen interactions on the nanopipette tip surface affect ionic current flowing through a 50-nm pore. Highly charged polymers interacting with the glass surface modulated the rectification property of the nanopipette electrode. Affinity-based binding between the probes tethered to the surface and their target proteins caused a change in the ionic current due to a partial blockade or an altered surface charge. These findings suggest that nanopipettes functionalized with appropriate molecular recognition elements can be used as nanosensors in biomedical and biological research.

  20. Targeting abnormal DNA double strand break repair in cancer

    OpenAIRE

    Rassool, Feyruz V.; Tomkinson, Alan E.

    2010-01-01

    A major challenge in cancer treatment is the development of therapies that target cancer cells with little or no toxicity to normal tissues and cells. Alterations in DNA double strand break (DSB) repair in cancer cells include both elevated and reduced levels of key repair proteins and changes in the relative contributions of the various DSB repair pathways. These differences can result in increased sensitivity to DSB-inducing agents and increased genomic instability. The development of agent...

  1. Targeting DNA repair systems in antitubercular drug development.

    Science.gov (United States)

    Minias, Alina; Brzostek, Anna; Dziadek, Jaroslaw

    2018-01-28

    Infections with Mycobacterium tuberculosis, the causative agent of tuberculosis, are difficult to treat using currently available chemotherapeutics. Clinicians agree on the urgent need for novel drugs to treat tuberculosis. In this mini review, we summarize data that prompts the consideration of DNA repair-associated proteins as targets for the development of new antitubercular compounds. We discuss data, including gene expression data, that highlight the importance of DNA repair genes during the pathogenic cycle as well as after exposure to antimicrobials currently in use. Specifically, we report experiments on determining the essentiality of DNA repair-related genes. We report the availability of protein crystal structures and summarize discovered protein inhibitors. Further, we describe phenotypes of available gene mutants of M. tuberculosis and model organisms Mycobacterium bovis and Mycobacterium smegmatis. We summarize experiments regarding the role of DNA repair-related proteins in pathogenesis and virulence performed both in vitro and in vivo during the infection of macrophages and animals. We detail the role of DNA repair genes in acquiring mutations, which influence the rate of drug resistance acquisition. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  2. Galactosylated DNA lipid nanocapsules for efficient hepatocyte targeting.

    Science.gov (United States)

    Morille, M; Passirani, C; Letrou-Bonneval, E; Benoit, J-P; Pitard, B

    2009-09-11

    The main objective of gene therapy via a systemic pathway is the development of a stable and non-toxic gene vector that can encapsulate and deliver foreign genetic materials into specific cell types with the transfection efficiency of viral vectors. With this objective, DNA complexed with cationic lipids of DOTAP/DOPE was encapsulated into lipid nanocapsules (LNCs) forming nanocarriers (DNA LNCs) with a size suitable for systemic injection (109+/-6 nm). With the goal of increasing systemic delivery, LNCs were stabilised with long chains of poly(ethylene glycol) (PEG), either from a PEG lipid derivative (DSPE-mPEG(2000)) or from an amphiphilic block copolymer (F108). In order to overcome internalisation difficulties encountered with PEG shield, a specific ligand (galactose) was covalently added at the distal end of the PEG chains, in order to provide active targeting of the asialoglycoprotein-receptor present on hepatocytes. This study showed that DNA LNCs were as efficient as positively charged DOTAP/DOPE lipoplexes for transfection. In primary hepatocytes, when non-galactosylated, the two polymers significantly decreased the transfection, probably by creating a barrier around the DNA LNCs. Interestingly, galactosylated F108 coated DNA LNCs led to a 18-fold increase in luciferase expression compared to non-galactosylated ones.

  3. Functionalized Polymer Microgel Particles Enable Customizable Production of Label-Free Sensor Arrays.

    Science.gov (United States)

    Lifson, Mark A; Carter, Jared A; Miller, Benjamin L

    2015-08-04

    Probe molecule immobilization onto surfaces is a critical step in the production of many analytical devices, including labeled and label-free microarrays. New methods to increase the density and uniformity of probe deposition have the potential to significantly enhance the ultimate limits of detection and reproducibility. Hydrogel-based materials have been employed in the past to provide a 3D protein-friendly surface for deposition of antibodies and nucleic acids. However, these methods are susceptible to variation during polymerization of the hydrogel scaffold and provide limited opportunities for tuning deposition parameters on an antibody-by-antibody basis. In this work, a versatile hydrogel nanoparticle deposition method was developed for the production of label-free microarrays and tested in the context of antibody-antigen binding. Poly(N-isopropylacrylamide) nanoparticles (PNIPAM) were conjugated to antibodies using an avidin/biotin system and deposited onto surfaces using a noncontact printing system. After drying, these gel spots formed uniform and thin layers <10 nm in height. The conjugates were characterized with dynamic light scattering, scanning electron microscopy, and atomic force microscopy. We tested this format in the context of tumor necrosis factor-alpha (TNF-α) detection via arrayed imaging reflectometry (AIR), a label-free protein microarray method. This method of probe molecule deposition should be generally useful in the production of microarrays for label-free detection.

  4. Toward microfluidic sperm refinement: continuous flow label-free analysis and sorting of sperm cells

    NARCIS (Netherlands)

    de Wagenaar, B.; Dekker, Stefan; van den Berg, Albert; Segerink, Loes Irene

    2015-01-01

    This manuscript reports upon the development of a microfluidic setup to detect and sort sperm cells from polystyrene beads label-free and non-invasively. Detection is performed by impedance analysis. When sperm cells passed the microelectrodes, the recorded impedance (19.6 ± 5.7 Ω) was higher

  5. Label-free SERS in biological and biomedical applications: Recent progress, current challenges and opportunities

    Science.gov (United States)

    Zheng, Xiao-Shan; Jahn, Izabella Jolan; Weber, Karina; Cialla-May, Dana; Popp, Jürgen

    2018-05-01

    To achieve an insightful look within biomolecular processes on the cellular level, the development of diseases as well as the reliable detection of metabolites and pathogens, a modern analytical tool is needed that is highly sensitive, molecular-specific and exhibits fast detection. Surface-enhanced Raman spectroscopy (SERS) is known to meet these requirements and, within this review article, the recent progress of label-free SERS in biological and biomedical applications is summarized and discussed. This includes the detection of biomolecules such as metabolites, nucleic acids and proteins. Further, the characterization and identification of microorganisms has been achieved by label-free SERS-based approaches. Eukaryotic cells can be characterized by SERS in order to gain information about the outer cell wall or to detect intracellular molecules and metabolites. The potential of SERS for medically relevant detection schemes is emphasized by the label-free detection of tissue, the investigation of body fluids as well as applications for therapeutic and illicit drug monitoring. The review article is concluded with an evaluation of the recent progress and current challenges in order to highlight the direction of label-free SERS in the future.

  6. Lab-on-a-chip for label free biological semiconductor analysis of Staphylococcal Enterotoxin B

    NARCIS (Netherlands)

    Yang, Minghui; Sun, Steven; Bruck, Hugh Alan; Kostov, Yordan; Rasooly, Avraham

    2010-01-01

    We describe a new lab-on-a-chip (LOC) which utilizes a biological semiconductor (BSC) transducer for label free analysis of Staphylococcal Enterotoxin B (SEB) (or other biological interactions) directly and electronically. BSCs are new transducers based on electrical percolation through a

  7. Scaffold-free, label-free and nozzle-free biofabrication technology using magnetic levitational assembly.

    Science.gov (United States)

    Parfenov, Vladislav A; Koudan, Elizaveta V; Bulanova, Elena A; Karalkin, Pavel A; Pereira, Frederico DAS; Norkin, Nikita E; Knyazeva, Alisa D; Gryadunova, Anna A; Petrov, Oleg F; Vasiliev, M M; Myasnikov, Maxim; Chernikov, Valery P; Kasyanov, Vladimir A; Marchenkov, Artem Yu; Brakke, Kenneth A; Khesuani, Yusef D; Demirci, Utkan; Mironov, Vladimir A

    2018-05-31

    Tissue spheroids have been proposed as building blocks in 3D biofabrication. Conventional magnetic force-driven 2D patterning of tissue spheroids requires prior cell labeling by magnetic nanoparticles, meanwhile a label-free approach for 3D magnetic levitational assembly has been introduced. Here we present first-time report on rapid assembly of 3D tissue construct using scaffold-free, nozzle-free and label-free magnetic levitation of tissue spheroids. Chondrospheres of standard size, shape and capable to fusion have been biofabricated from primary sheep chondrocytes using non-adhesive technology. Label-free magnetic levitation was performed using a prototype device equipped with permanent magnets in presence of gadolinium (Gd3+) in culture media, which enables magnetic levitation. Mathematical modeling and computer simulations were used for prediction of magnetic field and kinetics of tissue spheroids assembly into 3D tissue constructs. First, we used polystyrene beads to simulate the assembly of tissue spheroids and to determine the optimal settings for magnetic levitation in presence of Gd3+. Second, we proved the ability of chondrospheres to assemble rapidly into 3D tissue construct in the permanent magnetic field in the presence of Gd3+. Thus, scaffold- and label-free magnetic levitation of tissue spheroids is a promising approach for rapid 3D biofabrication and attractive alternative to label-based magnetic force-driven tissue engineering. . © 2018 IOP Publishing Ltd.

  8. Label-free biosensing with high sensitivity in dual-core microstructured polymer optical fibers

    DEFF Research Database (Denmark)

    Markos, Christos; Yuan, Wu; Vlachos, Kyriakos

    2011-01-01

    We present experimentally feasible designs of a dual-core microstructured polymer optical fiber (mPOF), which can act as a highly sensitive, label-free, and selective biosensor. An immobilized antigen sensing layer on the walls of the holes in the mPOF provides the ability to selectively capture...

  9. Label-free quantitative cell division monitoring of endothelial cells by digital holographic microscopy

    Science.gov (United States)

    Kemper, Björn; Bauwens, Andreas; Vollmer, Angelika; Ketelhut, Steffi; Langehanenberg, Patrik; Müthing, Johannes; Karch, Helge; von Bally, Gert

    2010-05-01

    Digital holographic microscopy (DHM) enables quantitative multifocus phase contrast imaging for nondestructive technical inspection and live cell analysis. Time-lapse investigations on human brain microvascular endothelial cells demonstrate the use of DHM for label-free dynamic quantitative monitoring of cell division of mother cells into daughter cells. Cytokinetic DHM analysis provides future applications in toxicology and cancer research.

  10. High throughput label-free platform for statistical bio-molecular sensing

    DEFF Research Database (Denmark)

    Bosco, Filippo; Hwu, En-Te; Chen, Ching-Hsiu

    2011-01-01

    Sensors are crucial in many daily operations including security, environmental control, human diagnostics and patient monitoring. Screening and online monitoring require reliable and high-throughput sensing. We report on the demonstration of a high-throughput label-free sensor platform utilizing...

  11. Polymer dual ring resonators for label-free optical biosensing using microfluidics.

    Science.gov (United States)

    Salleh, Muhammad H M; Glidle, Andrew; Sorel, Marc; Reboud, Julien; Cooper, Jonathan M

    2013-04-18

    We demonstrate a polymer resonator microfluidic biosensor that overcomes the complex manufacturing procedures required to fabricate traditional devices. In this new format, we show that a gapless light coupling photonic configuration, fabricated in SU8 polymer, can achieve high sensitivity, label-free chemical sensing in solution and high sensitivity biological sensing, at visible wavelengths.

  12. Rapid label-free profiling of oral cancer biomarker proteins using nano-UPLC-Q-TOF ion mobility mass spectrometry.

    Science.gov (United States)

    Nassar, Ala F; Williams, Brad J; Yaworksy, Dustin C; Patel, Vyomesh; Rusling, James F

    2016-03-01

    It has become quite clear that single cancer biomarkers cannot in general provide high sensitivity and specificity for reliable clinical cancer diagnostics. This paper explores the feasibility of rapid detection of multiple biomarker proteins in model oral cancer samples using label-free protein relative quantitation. MS-based label-free quantitative proteomics offer a rapid alternative that bypasses the need for stable isotope containing compounds to chemically bind and label proteins. Total protein content in oral cancer cell culture conditioned media was precipitated, subjected to proteolytic digestion, and then analyzed using a nano-UPLC (where UPLC is ultra-performance liquid chromatography) coupled to a hybrid Q-Tof ion-mobility mass spectrometry (MS). Rapid, simultaneous identification and quantification of multiple possible cancer biomarker proteins was achieved. In a comparative study between cancer and noncancer samples, approximately 952 proteins were identified using a high-throughput 1D ion mobility assisted data independent acquisition (IM-DIA) approach. As we previously demonstrated that interleukin-8 (IL-8) and vascular endothelial growth factor A (VEGF-A) were readily detected in oral cancer cell conditioned media(1), we targeted these biomarker proteins to validate our approach. Target biomarker protein IL-8 was found between 3.5 and 8.8 fmol, while VEGF-A was found at 1.45 fmol in the cancer cell media. Overall, our data suggest that the nano-UPLC-IM-DIA bioassay is a feasible approach to identify and quantify proteins in complex samples without the need for stable isotope labeling. These results have significant implications for rapid tumor diagnostics and prognostics by monitoring proteins such as IL-8 and VEGF-A implicated in cancer development and progression. The analysis in tissue or plasma is not possible at this time, but the subsequent work would be needed for validation. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Discovering the enzyme mimetic activity of metal-organic framework (MOF) for label-free and colorimetric sensing of biomolecules.

    Science.gov (United States)

    Wang, Ying; Zhu, Yingjing; Binyam, Atsebeha; Liu, Misha; Wu, Yinan; Li, Fengting

    2016-12-15

    A label-free sensing strategy based on the enzyme-mimicking activity of MOF was demonstrated for colorimetric detection of biomolecules. Firstly obvious blue color was observed due to the high efficiency of peroxidase-like catalytic activity of Fe-MIL-88A (an ion-based MOF material) toward 3,3',5,5'-tetramethylbenzidine (TMB). Then in the presence of target biomolecule and corresponding aptamer, the mimetic activity of Fe-MIL-88A can be strongly inhibited and used directly to realize the colorimetric detection. On the basis of the interesting findings, we designed a straightforward, label-free and sensitive colorimetric method for biomolecule detection by using the enzyme mimetic property of MOF coupling with molecular recognition element. Compared with the existed publications, our work breaks the routine way by setting up an inorganic-organic MOF-aptamer hybrid platform for colorimetric determination of biomolecules, expanding the targets scope from H2O2 or glucose to biomolecules. As a proof of concept, thrombin and thrombin aptamer was used as a model analyte. The limit of detection of 10nM can be achieved with naked eyes and ultrahigh selectivity of thrombin toward numerous interfering substances with 10-fold concentration was demonstrated significantly. Of note, the method was further applied for the detection of thrombin in human serum samples, showing the results in agreement with those values obtained in an immobilization buffer by the colorimetric method. This inorganic-organic MOF-aptamer sensing strategy may in principle be universally applicable for the detection of a range of environmental or biomedical molecules of interests. Copyright © 2016 Elsevier B.V. All rights reserved.

  14. Auger radiation targeted into DNA: a therapy perspective

    International Nuclear Information System (INIS)

    Buchegger, Franz; Perillo-Adamer, Florence; Bischof Delaloye, Angelika; Dupertuis, Yves M.

    2006-01-01

    Auger electron emitters that can be targeted into DNA of tumour cells represent an attractive systemic radiation therapy goal. In the situation of DNA-associated decay, the high linear energy transfer (LET) of Auger electrons gives a high relative biological efficacy similar to that of α particles. In contrast to α radiation, however, Auger radiation is of low toxicity when decaying outside the cell nucleus, as in cytoplasm or outside cells during blood transport. The challenge for such therapies is the requirement to target a high percentage of all cancer cells. An overview of Auger radiation therapy approaches of the past decade shows several research directions and various targeting vehicles. The latter include hormones, peptides, halogenated nucleotides, oligonucleotides and internalising antibodies. Here, we will discuss the basic principles of Auger electron therapy as compared with vector-guided α and β radiation. We also review some radioprotection issues and briefly present the main advantages and disadvantages of the different targeting modalities that are under investigation. (orig.)

  15. Auger radiation targeted into DNA: a therapy perspective

    Energy Technology Data Exchange (ETDEWEB)

    Buchegger, Franz [University Hospital of Lausanne CHUV, Service of Nuclear Medicine, Lausanne (Switzerland); University Hospital of Lausanne, Service of Nuclear Medicine, Lausanne (Switzerland); Perillo-Adamer, Florence; Bischof Delaloye, Angelika [University Hospital of Lausanne CHUV, Service of Nuclear Medicine, Lausanne (Switzerland); Dupertuis, Yves M. [University Hospital of Geneva, Service of Nutrition, Geneva (Switzerland)

    2006-11-15

    Auger electron emitters that can be targeted into DNA of tumour cells represent an attractive systemic radiation therapy goal. In the situation of DNA-associated decay, the high linear energy transfer (LET) of Auger electrons gives a high relative biological efficacy similar to that of {alpha} particles. In contrast to {alpha} radiation, however, Auger radiation is of low toxicity when decaying outside the cell nucleus, as in cytoplasm or outside cells during blood transport. The challenge for such therapies is the requirement to target a high percentage of all cancer cells. An overview of Auger radiation therapy approaches of the past decade shows several research directions and various targeting vehicles. The latter include hormones, peptides, halogenated nucleotides, oligonucleotides and internalising antibodies. Here, we will discuss the basic principles of Auger electron therapy as compared with vector-guided {alpha} and {beta} radiation. We also review some radioprotection issues and briefly present the main advantages and disadvantages of the different targeting modalities that are under investigation. (orig.)

  16. DNA-Destabilizing Agents as an Alternative Approach for Targeting DNA: Mechanisms of Action and Cellular Consequences

    Directory of Open Access Journals (Sweden)

    Gaëlle Lenglet

    2010-01-01

    Full Text Available DNA targeting drugs represent a large proportion of the actual anticancer drug pharmacopeia, both in terms of drug brands and prescription volumes. Small DNA-interacting molecules share the ability of certain proteins to change the DNA helix's overall organization and geometrical orientation via tilt, roll, twist, slip, and flip effects. In this ocean of DNA-interacting compounds, most stabilize both DNA strands and very few display helix-destabilizing properties. These types of DNA-destabilizing effect are observed with certain mono- or bis-intercalators and DNA alkylating agents (some of which have been or are being developed as cancer drugs. The formation of locally destabilized DNA portions could interfere with protein/DNA recognition and potentially affect several crucial cellular processes, such as DNA repair, replication, and transcription. The present paper describes the molecular basis of DNA destabilization, the cellular impact on protein recognition, and DNA repair processes and the latter's relationships with antitumour efficacy.

  17. Patterned Array of Poly(ethylene glycol Silane Monolayer for Label-Free Detection of Dengue

    Directory of Open Access Journals (Sweden)

    Nor Zida Rosly

    2016-08-01

    Full Text Available In the present study, the construction of arrays on silicon for naked-eye detection of DNA dengue was demonstrated. The array was created by exposing a polyethylene glycol (PEG silane monolayer to 254 nm ultraviolet (UV light through a photomask. Formation of the PEG silane monolayer and photomodifed surface properties was thoroughly characterized by using atomic force microscopy (AFM, X-ray photoelectron spectroscopy (XPS, and contact angle measurements. The results of XPS confirmed that irradiation of ultraviolet (UV light generates an aldehyde functional group that offers conjugation sites of amino DNA probe for detection of a specific dengue virus target DNA. Employing a gold enhancement process after inducing the electrostatic interaction between positively charged gold nanoparticles and the negatively charged target DNA hybridized to the DNA capture probe allowed to visualize the array with naked eye. The developed arrays demonstrated excellent performance in diagnosis of dengue with a detection limit as low as 10 pM. The selectivity of DNA arrays was also examined using a single base mismatch and noncomplementary target DNA.

  18. Label-free super sandwich electrogenerated chemiluminescence biosensor for the determination of the HIV gene

    International Nuclear Information System (INIS)

    Ruan, Sanpeng; Li, Zhejian; Qi, Honglan; Gao, Qiang; Zhang, Chengxiao

    2014-01-01

    We describe a highly sensitive electrochemiluminescence (ECL) based method for the determination of the human immunodeficiency virus-1 (HIV-1) gene. A long-range self-assembled double strand DNA (ds-DNA) is used as a carrier, and the ruthenium complex Ru(phen) 3 2+ as an ECL indicator for signal amplification. The thiolated ss-DNA serving as a capture probe is firstly self-assembled on the surface of a gold electrode. After the target HIV-1 gene is completely hybridized with the capture probe, two previously hybridized auxiliary probes are hybridized with the target HIV-1 gene to form long-range super sandwich ds-DNA polymers on the surface of the electrode. Finally, the ECL indicator is intercalated into the super sandwich ds-DNA grooves. This results in a strongly increased ECL in tripropylamine solution because a large fraction of the intercalator is intercalated into super sandwich ds-DNA. The results showed that the increased ECL intensity is directly related to the logarithm of the concentration of the HIV-1 gene in the range from 0.1 pM to 0.1 nM, with a detection limit of 0.022 pM and using only 10 μL of analyte samples. The method can effectively discriminate target HIV-1 gene (a perfectly matched ss-DNA) from a 2-base mismatched ss-DNA. This work demonstrates that the high sensitivity and selectivity of an ECL DNA biosensor can be largely improved by using super sandwich ds-DNA along with ECL indicators. (author)

  19. A regenerated electrochemical biosensor for label-free detection of glucose and urea based on conformational switch of i-motif oligonucleotide probe

    Energy Technology Data Exchange (ETDEWEB)

    Gao, Zhong Feng; Chen, Dong Mei [Key Laboratory of Eco-environments in Three Gorges Reservoir Region (Ministry of Education), School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715 (China); Lei, Jing Lei [School of Chemistry and Chemical Engineering, Chongqing University, Chongqing 400044 (China); Luo, Hong Qun, E-mail: luohq@swu.edu.cn [Key Laboratory of Eco-environments in Three Gorges Reservoir Region (Ministry of Education), School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715 (China); Li, Nian Bing, E-mail: linb@swu.edu.cn [Key Laboratory of Eco-environments in Three Gorges Reservoir Region (Ministry of Education), School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715 (China)

    2015-10-15

    Improving the reproducibility of electrochemical signal remains a great challenge over the past decades. In this work, i-motif oligonucleotide probe-based electrochemical DNA (E-DNA) sensor is introduced for the first time as a regenerated sensing platform, which enhances the reproducibility of electrochemical signal, for label-free detection of glucose and urea. The addition of glucose or urea is able to activate glucose oxidase-catalyzed or urease-catalyzed reaction, inducing or destroying the formation of i-motif oligonucleotide probe. The conformational switch of oligonucleotide probe can be recorded by electrochemical impedance spectroscopy. Thus, the difference of electron transfer resistance is utilized for the quantitative determination of glucose and urea. We further demonstrate that the E-DNA sensor exhibits high selectivity, excellent stability, and remarkable regenerated ability. The human serum analysis indicates that this simple and regenerated strategy holds promising potential in future biosensing applications. - Highlights: • Conformational switch of i-motif is used for the detection of glucose and urea. • The sensor can be regenerated. • The proposed method is successfully applied in real sample assay. • Our method is label-free and inexpensive.

  20. Molecular targets, DNA breakage, DNA repair: Their roles in mutation induction in mammalian germ cells

    International Nuclear Information System (INIS)

    Sega, G.A.

    1989-01-01

    Variability in genetic sensitivity among different germ-cell stages in the mammal to various mutagens could be the result of how much chemical reaches the different stages, what molecular targets may be affected in the different stages and whether or not repair of lesions occurs. Several chemicals have been found to bind very strongly to protamine in late-spermatid and early-spermatozoa stages in the mouse. The chemicals also produce their greatest genetic damage in these same germ-cell stages. While chemical binding to DNA has not been correlated with the level of induced genetic damage, DNA breakage in the sensitive stages has been shown to increase. This DNA breakage is believed to indirectly result from chemical binding to sulfhydryl groups in protamine which prevents normal chromatin condensation within the sperm nucleus. 22 refs., 5 figs

  1. Multifunctional DNA-gold nanoparticles for targeted doxorubicin delivery.

    Science.gov (United States)

    Alexander, Colleen M; Hamner, Kristen L; Maye, Mathew M; Dabrowiak, James C

    2014-07-16

    In this report we describe the synthesis, characterization, and cytotoxic properties of DNA-capped gold nanoparticles having attached folic acid (FA), a thermoresponsive polymer (p), and/or poly(ethylene glycol) (PEG) oligomers that could be used to deliver the anticancer drug doxorubicin (DOX) in chemotherapy. The FA-DNA oligomer used in the construction of the delivery vehicle was synthesized through the reaction of the isolated folic acid N-hydroxysuccinimide ester with the amino-DNA and the conjugated DNA product was purified using high performance liquid chromatography (HPLC). This approach ultimately allowed control of the amount of FA attached to the surface of the delivery vehicle. Cytotoxicity studies using SK-N-SH neuroblastoma cells with drug loaded delivery vehicles were carried out using a variety of exposure times (1-48 h) and recovery times (1-72 h), and in order to access the effects of varying amounts of attached FA, in culture media deficient in FA. DOX loaded delivery vehicles having 50% of the DNA strands with attached FA were more cytotoxic than when all of the strands contained FA. Since FA stimulates cell growth, the reduced cytotoxicity of vehicles fully covered with FA suggests that the stimulatory effects of FA can more than compensate for the cytotoxic effects of the drug on the cell population. While attachment of hexa-ethylene glycol PEG(18) to the surface of the delivery vehicle had no effect on cytotoxicity, 100% FA plus the thermoresponsive polymer resulted in IC50 = 0.48 ± 0.01 for an exposure time of 24 h and a recovery time of 1 h, which is an order of magnitude more cytotoxic than free DOX. Confocal microscopic studies using fluorescence detection showed that SK-N-SH neuroblastoma cells exposed to DOX-loaded vehicles have drug accumulation inside the cell and, in the case of vehicles with attached FA and thermoresponsive polymer, the drug appears more concentrated. Since the biological target of DOX is DNA, the latter

  2. Highly sensitive and label-free electrochemical detection of microRNAs based on triple signal amplification of multifunctional gold nanoparticles, enzymes and redox-cycling reaction.

    Science.gov (United States)

    Liu, Lin; Xia, Ning; Liu, Huiping; Kang, Xiaojing; Liu, Xiaoshuan; Xue, Chan; He, Xiaoling

    2014-03-15

    MicroRNAs (miRNAs) are believed to be important for cancer diagnosis and prognosis, serving as reliable molecular biomarkers. In this work, we presented a label-free and highly sensitive electrochemical genosensor for miRNAs detection with the triple signal amplification of gold nanoparticles (AuNPs), alkaline phosphatase (ALP) and p-aminophenol (p-AP) redox cycling. The label-free strategy is based on the difference in the structures of RNA and DNA. Specifically, miRNAs were first captured by the pre-immobilized DNA probes on a gold electrode. Next, the cis-diol group of ribose sugar at the end of the miRNAs chain allowed 3-aminophenylboronic acid (APBA)/biotin-modified multifunctional AuNPs (denoted as APBA-biotin-AuNPs) to be attached through the formation of a boronate ester covalent bond, which facilitated the capture of streptavidin-conjugated alkaline phosphatase (SA-ALP) via the biotin-streptavidin interaction. After the addition of the 4-aminophenylphosphate (p-APP) substrate, the enzymatic conversion from p-APP to p-AP occurred. The resulting p-AP could be cycled by a chemical reducing reagent after its electro-oxidization on the electrode (known as p-AP redox cycling), thus enabling an increase in the anodic current. As a result, the current increased linearly with the miRNAs concentration over a range of 10 fM-5 pM, and a detection limit of 3 fM was achieved. We believe that this work will be valuable for the design of new types of label-free and sensitive electrochemical biosensors. © 2013 Published by Elsevier B.V.

  3. Label free imaging of cell-substrate contacts by holographic total internal reflection microscopy.

    Science.gov (United States)

    Mandracchia, Biagio; Gennari, Oriella; Marchesano, Valentina; Paturzo, Melania; Ferraro, Pietro

    2017-09-01

    The study of cell adhesion contacts is pivotal to understand cell mechanics and interaction at substrates or chemical and physical stimuli. We designed and built a HoloTIR microscope for label-free quantitative phase imaging of total internal reflection. Here we show for the first time that HoloTIR is a good choice for label-free study of focal contacts and of cell/substrate interaction as its sensitivity is enhanced in comparison with standard TIR microscopy. Finally, the simplicity of implementation and relative low cost, due to the requirement of less optical components, make HoloTIR a reasonable alternative, or even an addition, to TIRF microscopy for mapping cell/substratum topography. As a proof of concept, we studied the formation of focal contacts of fibroblasts on three substrates with different levels of affinity for cell adhesion. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Label-Free Electrical Detection Using Carbon Nanotube-Based Biosensors

    Directory of Open Access Journals (Sweden)

    Kenzo Maehashi

    2009-07-01

    Full Text Available Label-free detections of biomolecules have attracted great attention in a lot of life science fields such as genomics, clinical diagnosis and practical pharmacy. In this article, we reviewed amperometric and potentiometric biosensors based on carbon nanotubes (CNTs. In amperometric detections, CNT-modified electrodes were used as working electrodes to significantly enhance electroactive surface area. In contrast, the potentiometric biosensors were based on aptamer-modified CNT field-effect transistors (CNTFETs. Since aptamers are artificial oligonucleotides and thus are smaller than the Debye length, proteins can be detected with high sensitivity. In this review, we discussed on the technology, characteristics and developments for commercialization in label-free CNT-based biosensors.

  5. Artificial intelligence in label-free microscopy biological cell classification by time stretch

    CERN Document Server

    Mahjoubfar, Ata; Jalali, Bahram

    2017-01-01

    This book introduces time-stretch quantitative phase imaging (TS-QPI), a high-throughput label-free imaging flow cytometer developed for big data acquisition and analysis in phenotypic screening. TS-QPI is able to capture quantitative optical phase and intensity images simultaneously, enabling high-content cell analysis, cancer diagnostics, personalized genomics, and drug development. The authors also demonstrate a complete machine learning pipeline that performs optical phase measurement, image processing, feature extraction, and classification, enabling high-throughput quantitative imaging that achieves record high accuracy in label -free cellular phenotypic screening and opens up a new path to data-driven diagnosis. • Demonstrates how machine learning is used in high-speed microscopy imaging to facilitate medical diagnosis; • Provides a systematic and comprehensive illustration of time stretch technology; • Enables multidisciplinary application, including industrial, biomedical, and artificial intell...

  6. Label-free and live cell imaging by interferometric scattering microscopy.

    Science.gov (United States)

    Park, Jin-Sung; Lee, Il-Buem; Moon, Hyeon-Min; Joo, Jong-Hyeon; Kim, Kyoung-Hoon; Hong, Seok-Cheol; Cho, Minhaeng

    2018-03-14

    Despite recent remarkable advances in microscopic techniques, it still remains very challenging to directly observe the complex structure of cytoplasmic organelles in live cells without a fluorescent label. Here we report label-free and live-cell imaging of mammalian cell, Escherischia coli , and yeast, using interferometric scattering microscopy, which reveals the underlying structures of a variety of cytoplasmic organelles as well as the underside structure of the cells. The contact areas of the cells attached onto a glass substrate, e.g. , focal adhesions and filopodia, are clearly discernible. We also found a variety of fringe-like features in the cytoplasmic area, which may reflect the folded structures of cytoplasmic organelles. We thus anticipate that the label-free interferometric scattering microscopy can be used as a powerful tool to shed interferometric light on in vivo structures and dynamics of various intracellular phenomena.

  7. Robust label-free biosensing using microdisk laser arrays with on-chip references.

    Science.gov (United States)

    Wondimu, S F; Hippler, M; Hussal, C; Hofmann, A; Krämmer, S; Lahann, J; Kalt, H; Freude, W; Koos, C

    2018-02-05

    Whispering-gallery mode (WGM) microdisk lasers show great potential for highly sensitive label-free detection in large-scale sensor arrays. However, when used in practical applications under normal ambient conditions, these devices suffer from temperature fluctuations and photobleaching. Here we demonstrate that these challenges can be overcome by a novel referencing scheme that allows for simultaneous compensation of temperature drift and photobleaching. The technique relies on reference structures protected by locally dispensed passivation materials, and can be scaled to extended arrays of hundreds of devices. We prove the viability of the concept in a series of experiments, demonstrating robust and sensitive label-free detection over a wide range of constant or continuously varying temperatures. To the best of our knowledge, these measurements represent the first demonstration of biosensing in active WGM devices with simultaneous compensation of both photobleaching and temperature drift.

  8. Label-Free Aptasensor for Lysozyme Detection Using Electrochemical Impedance Spectroscopy

    OpenAIRE

    Dionisia Ortiz-Aguayo; Manel del Valle

    2018-01-01

    This research develops a label-free aptamer biosensor (aptasensor) based on graphite-epoxy composite electrodes (GECs) for the detection of lysozyme protein using Electrochemical Impedance Spectroscopy (EIS) technique. The chosen immobilization technique was based on covalent bonding using carbodiimide chemistry; for this purpose, carboxylic moieties were first generated on the graphite by electrochemical grafting. The detection was performed using [Fe(CN)6]3−/[Fe(CN)6]4− as redox probe. Afte...

  9. Optimization of High-Q Coupled Nanobeam Cavity for Label-Free Sensing

    OpenAIRE

    Yaseen, Mohammad; Yang, Yi-Chun; Shih, Min-Hsiung; Chang, Yia-Chung

    2015-01-01

    We numerically and experimentally investigated the lateral coupling between photonic crystal (PhC) nanobeam (NB) cavities, pursuing high sensitivity and figure of merit (FOM) label-free biosensor. We numerically carried out 3D finite-difference time-domain (3D-FDTD) and the finite element method (FEM) simulations. We showed that when two PhC NB cavities separated by a small gap are evanescently coupled, the variation in the gap width significantly changes the coupling efficiency between the ...

  10. Label-Free Bioanalyte Detection from Nanometer to Micrometer Dimensions—Molecular Imprinting and QCMs †

    Directory of Open Access Journals (Sweden)

    Adnan Mujahid

    2018-06-01

    Full Text Available Modern diagnostic tools and immunoassay protocols urges direct analyte recognition based on its intrinsic behavior without using any labeling indicator. This not only improves the detection reliability, but also reduces sample preparation time and complexity involved during labeling step. Label-free biosensor devices are capable of monitoring analyte physiochemical properties such as binding sensitivity and selectivity, affinity constants and other dynamics of molecular recognition. The interface of a typical biosensor could range from natural antibodies to synthetic receptors for example molecular imprinted polymers (MIPs. The foremost advantages of using MIPs are their high binding selectivity comparable to natural antibodies, straightforward synthesis in short time, high thermal/chemical stability and compatibility with different transducers. Quartz crystal microbalance (QCM resonators are leading acoustic devices that are extensively used for mass-sensitive measurements. Highlight features of QCM devices include low cost fabrication, room temperature operation, and most importantly ability to monitor extremely low mass shifts, thus potentially a universal transducer. The combination of MIPs with quartz QCM has turned out as a prominent sensing system for label-free recognition of diverse bioanalytes. In this article, we shall encompass the potential applications of MIP-QCM sensors exclusively label-free recognition of bacteria and virus species as representative micro and nanosized bioanalytes.

  11. A multi-center study benchmarks software tools for label-free proteome quantification

    Science.gov (United States)

    Gillet, Ludovic C; Bernhardt, Oliver M.; MacLean, Brendan; Röst, Hannes L.; Tate, Stephen A.; Tsou, Chih-Chiang; Reiter, Lukas; Distler, Ute; Rosenberger, George; Perez-Riverol, Yasset; Nesvizhskii, Alexey I.; Aebersold, Ruedi; Tenzer, Stefan

    2016-01-01

    The consistent and accurate quantification of proteins by mass spectrometry (MS)-based proteomics depends on the performance of instruments, acquisition methods and data analysis software. In collaboration with the software developers, we evaluated OpenSWATH, SWATH2.0, Skyline, Spectronaut and DIA-Umpire, five of the most widely used software methods for processing data from SWATH-MS (sequential window acquisition of all theoretical fragment ion spectra), a method that uses data-independent acquisition (DIA) for label-free protein quantification. We analyzed high-complexity test datasets from hybrid proteome samples of defined quantitative composition acquired on two different MS instruments using different SWATH isolation windows setups. For consistent evaluation we developed LFQbench, an R-package to calculate metrics of precision and accuracy in label-free quantitative MS, and report the identification performance, robustness and specificity of each software tool. Our reference datasets enabled developers to improve their software tools. After optimization, all tools provided highly convergent identification and reliable quantification performance, underscoring their robustness for label-free quantitative proteomics. PMID:27701404

  12. A multicenter study benchmarks software tools for label-free proteome quantification.

    Science.gov (United States)

    Navarro, Pedro; Kuharev, Jörg; Gillet, Ludovic C; Bernhardt, Oliver M; MacLean, Brendan; Röst, Hannes L; Tate, Stephen A; Tsou, Chih-Chiang; Reiter, Lukas; Distler, Ute; Rosenberger, George; Perez-Riverol, Yasset; Nesvizhskii, Alexey I; Aebersold, Ruedi; Tenzer, Stefan

    2016-11-01

    Consistent and accurate quantification of proteins by mass spectrometry (MS)-based proteomics depends on the performance of instruments, acquisition methods and data analysis software. In collaboration with the software developers, we evaluated OpenSWATH, SWATH 2.0, Skyline, Spectronaut and DIA-Umpire, five of the most widely used software methods for processing data from sequential window acquisition of all theoretical fragment-ion spectra (SWATH)-MS, which uses data-independent acquisition (DIA) for label-free protein quantification. We analyzed high-complexity test data sets from hybrid proteome samples of defined quantitative composition acquired on two different MS instruments using different SWATH isolation-window setups. For consistent evaluation, we developed LFQbench, an R package, to calculate metrics of precision and accuracy in label-free quantitative MS and report the identification performance, robustness and specificity of each software tool. Our reference data sets enabled developers to improve their software tools. After optimization, all tools provided highly convergent identification and reliable quantification performance, underscoring their robustness for label-free quantitative proteomics.

  13. freeQuant: A Mass Spectrometry Label-Free Quantification Software Tool for Complex Proteome Analysis.

    Science.gov (United States)

    Deng, Ning; Li, Zhenye; Pan, Chao; Duan, Huilong

    2015-01-01

    Study of complex proteome brings forward higher request for the quantification method using mass spectrometry technology. In this paper, we present a mass spectrometry label-free quantification tool for complex proteomes, called freeQuant, which integrated quantification with functional analysis effectively. freeQuant consists of two well-integrated modules: label-free quantification and functional analysis with biomedical knowledge. freeQuant supports label-free quantitative analysis which makes full use of tandem mass spectrometry (MS/MS) spectral count, protein sequence length, shared peptides, and ion intensity. It adopts spectral count for quantitative analysis and builds a new method for shared peptides to accurately evaluate abundance of isoforms. For proteins with low abundance, MS/MS total ion count coupled with spectral count is included to ensure accurate protein quantification. Furthermore, freeQuant supports the large-scale functional annotations for complex proteomes. Mitochondrial proteomes from the mouse heart, the mouse liver, and the human heart were used to evaluate the usability and performance of freeQuant. The evaluation showed that the quantitative algorithms implemented in freeQuant can improve accuracy of quantification with better dynamic range.

  14. Accurate label-free reaction kinetics determination using initial rate heat measurements

    Science.gov (United States)

    Ebrahimi, Kourosh Honarmand; Hagedoorn, Peter-Leon; Jacobs, Denise; Hagen, Wilfred R.

    2015-01-01

    Accurate label-free methods or assays to obtain the initial reaction rates have significant importance in fundamental studies of enzymes and in application-oriented high throughput screening of enzyme activity. Here we introduce a label-free approach for obtaining initial rates of enzyme activity from heat measurements, which we name initial rate calorimetry (IrCal). This approach is based on our new finding that the data recorded by isothermal titration calorimetry for the early stages of a reaction, which have been widely ignored, are correlated to the initial rates. Application of the IrCal approach to various enzymes led to accurate enzyme kinetics parameters as compared to spectroscopic methods and enabled enzyme kinetic studies with natural substrate, e.g. proteases with protein substrates. Because heat is a label-free property of almost all reactions, the IrCal approach holds promise in fundamental studies of various enzymes and in use of calorimetry for high throughput screening of enzyme activity. PMID:26574737

  15. Nanoplasmonic biochips for rapid label-free detection of imidacloprid pesticides with a smartphone.

    Science.gov (United States)

    Lee, Kuang-Li; You, Meng-Lin; Tsai, Chia-Hsin; Lin, En-Hung; Hsieh, Shu-Yi; Ho, Ming-Hsun; Hsu, Ju-Chun; Wei, Pei-Kuen

    2016-01-15

    The widespread and intensive use of neonicotinoid insecticides induces negative cascading effects on ecosystems. It is desirable to develop a portable sensitive sensing platform for on-site screening of high-risk pesticides. We combined an indirect competitive immunoassay, highly sensitive surface plasmon resonance (SPR) biochip and a simple portable imaging setup for label-free detection of imidacloprid pesticides. The SPR biochip consists of several capped nanoslit arrays with different periods which form a spectral image on the chip. The qualitative and semiquantitative analyses of pesticides can be directly observed from the spot shift on the chip. The precise semiquantitative analyses can be further completed by using image processing in a smartphone. We demonstrate simultaneous detection of four different concentrations of imidacloprid pesticides. The visual detection limit is about 1ppb, which is well below the maximum residue concentration permitted by law (20ppb). Compared to the one-step strip assay, the proposed chip is capable of performing semiquantitative analyses and multiple detection. Compared to the enzyme-linked immunosorbent assay, our method is label-free and requires simple washing steps and short reaction time. In addition, the label-free chip has a comparable sensitivity but wider working range than those labeling techniques. Copyright © 2015 Elsevier B.V. All rights reserved.

  16. Multimodal nonlinear microscopy: A powerful label-free method for supporting standard diagnostics on biological tissues

    Directory of Open Access Journals (Sweden)

    Riccardo Cicchi

    2014-09-01

    Full Text Available The large use of nonlinear laser scanning microscopy in the past decade paved the way for potential clinical application of this imaging technique. Modern nonlinear microscopy techniques offer promising label-free solutions to improve diagnostic performances on tissues. In particular, the combination of multiple nonlinear imaging techniques in the same microscope allows integrating morphological with functional information in a morpho-functional scheme. Such approach provides a high-resolution label-free alternative to both histological and immunohistochemical examination of tissues and is becoming increasingly popular among the clinical community. Nevertheless, several technical improvements, including automatic scanning and image analysis, are required before the technique represents a standard diagnostic method. In this review paper, we highlight the capabilities of multimodal nonlinear microscopy for tissue imaging, by providing various examples on colon, arterial and skin tissues. The comparison between images acquired using multimodal nonlinear microscopy and histology shows a good agreement between the two methods. The results demonstrate that multimodal nonlinear microscopy is a powerful label-free alternative to standard histopathological methods and has the potential to find a stable place in the clinical setting in the near future.

  17. Label-Free Alignment of Nonmagnetic Particles in a Small Uniform Magnetic Field.

    Science.gov (United States)

    Wang, Zhaomeng; Wang, Ying; Wu, Rui Ge; Wang, Z P; Ramanujan, R V

    2018-01-01

    Label-free manipulation of biological entities can minimize damage, increase viability and improve efficiency of subsequent analysis. Understanding the mechanism of interaction between magnetic and nonmagnetic particles in an inverse ferrofluid can provide a mechanism of label-free manipulation of such entities in a uniform magnetic field. The magnetic force, induced by relative magnetic susceptibility difference between nonmagnetic particles and surrounding magnetic particles as well as particle-particle interaction were studied. Label-free alignment of nonmagnetic particles can be achieved by higher magnetic field strength (Ba), smaller particle spacing (R), larger particle size (rp1), and higher relative magnetic permeability difference between particle and the surrounding fluid (Rμr). Rμr can be used to predict the direction of the magnetic force between both magnetic and nonmagnetic particles. A sandwich structure, containing alternate layers of magnetic and nonmagnetic particle chains, was studied. This work can be used for manipulation of nonmagnetic particles in lab-on-a-chip applications.

  18. Solution-based targeted genomic enrichment for precious DNA samples

    Directory of Open Access Journals (Sweden)

    Shearer Aiden

    2012-05-01

    Full Text Available Abstract Background Solution-based targeted genomic enrichment (TGE protocols permit selective sequencing of genomic regions of interest on a massively parallel scale. These protocols could be improved by: 1 modifying or eliminating time consuming steps; 2 increasing yield to reduce input DNA and excessive PCR cycling; and 3 enhancing reproducible. Results We developed a solution-based TGE method for downstream Illumina sequencing in a non-automated workflow, adding standard Illumina barcode indexes during the post-hybridization amplification to allow for sample pooling prior to sequencing. The method utilizes Agilent SureSelect baits, primers and hybridization reagents for the capture, off-the-shelf reagents for the library preparation steps, and adaptor oligonucleotides for Illumina paired-end sequencing purchased directly from an oligonucleotide manufacturing company. Conclusions This solution-based TGE method for Illumina sequencing is optimized for small- or medium-sized laboratories and addresses the weaknesses of standard protocols by reducing the amount of input DNA required, increasing capture yield, optimizing efficiency, and improving reproducibility.

  19. The proteome of human liver peroxisomes: identification of five new peroxisomal constituents by a label-free quantitative proteomics survey.

    Directory of Open Access Journals (Sweden)

    Thomas Gronemeyer

    Full Text Available The peroxisome is a key organelle of low abundance that fulfils various functions essential for human cell metabolism. Severe genetic diseases in humans are caused by defects in peroxisome biogenesis or deficiencies in the function of single peroxisomal proteins. To improve our knowledge of this important cellular structure, we studied for the first time human liver peroxisomes by quantitative proteomics. Peroxisomes were isolated by differential and Nycodenz density gradient centrifugation. A label-free quantitative study of 314 proteins across the density gradient was accomplished using high resolution mass spectrometry. By pairing statistical data evaluation, cDNA cloning and in vivo colocalization studies, we report the association of five new proteins with human liver peroxisomes. Among these, isochorismatase domain containing 1 protein points to the existence of a new metabolic pathway and hydroxysteroid dehydrogenase like 2 protein is likely involved in the transport or β-oxidation of fatty acids in human peroxisomes. The detection of alcohol dehydrogenase 1A suggests the presence of an alternative alcohol-oxidizing system in hepatic peroxisomes. In addition, lactate dehydrogenase A and malate dehydrogenase 1 partially associate with human liver peroxisomes and enzyme activity profiles support the idea that NAD(+ becomes regenerated during fatty acid β-oxidation by alternative shuttling processes in human peroxisomes involving lactate dehydrogenase and/or malate dehydrogenase. Taken together, our data represent a valuable resource for future studies of peroxisome biochemistry that will advance research of human peroxisomes in health and disease.

  20. Selection of specific aptamer against enrofloxacin and fabrication of graphene oxide based label-free fluorescent assay.

    Science.gov (United States)

    Dolati, Somayeh; Ramezani, Mohammad; Nabavinia, Maryam Sadat; Soheili, Vahid; Abnous, Khalil; Taghdisi, Seyed Mohammad

    2018-05-15

    Specific ssDNA aptamers for the antibiotic enrofloxacin (ENR) were isolated from an enriched nucleotide library by SELEX (Systematic Evolution of Ligands by EXponential enrichment) method with high binding affinity. After seven rounds, five aptamers were selected and identified. Apt58 with highest affinity and sensitivity (K d  = 14.19 nM) was employed to develop a label-free fluorescent biosensing approach based on aptamer, graphene oxide (GO) and native fluorescence of ENR for determination of ENR residue in raw milk samples. Under optimized experimental conditions, the linear range was from 5 nM to 250 nM and LOD was calculated to be 3.7 nM, and the recovery rate was between 94.1% and 108.5%. The integration of aptamer and GO in this bioassay provides a promising way for rapid, sensitive and cost-effective detection of ENR in real samples like raw milk. Copyright © 2018 Elsevier Inc. All rights reserved.

  1. Quantitative evaluation of alternatively spliced mRNA isoforms by label-free real-time plasmonic sensing.

    Science.gov (United States)

    Huertas, César S; Carrascosa, L G; Bonnal, S; Valcárcel, J; Lechuga, L M

    2016-04-15

    Alternative splicing of mRNA precursors enables cells to generate different protein outputs from the same gene depending on their developmental or homeostatic status. Its deregulation is strongly linked to disease onset and progression. Current methodologies for monitoring alternative splicing demand elaborate procedures and often present difficulties in discerning between closely related isoforms, e.g. due to cross-hybridization during their detection. Herein, we report a general methodology using a Surface Plasmon Resonance (SPR) biosensor for label-free monitoring of alternative splicing events in real-time, without any cDNA synthesis or PCR amplification requirements. We applied this methodology to RNA isolated from HeLa cells for the quantification of alternatively spliced isoforms of the Fas gene, involved in cancer progression through regulation of programmed cell death. We demonstrate that our methodology is isoform-specific, with virtually no cross-hybridization, achieving limits of detection (LODs) in the picoMolar (pM) range. Similar results were obtained for the detection of the BCL-X gene mRNA isoforms. The results were independently validated by RT-qPCR, with excellent concordance in the determination of isoform ratios. The simplicity and robustness of this biosensor technology can greatly facilitate the exploration of alternative splicing biomarkers in disease diagnosis and therapy. Copyright © 2015 Elsevier B.V. All rights reserved.

  2. Label free targeted detection and quantification of celiac disease immunogenic epitopes by mass spectrometry

    NARCIS (Netherlands)

    Broeck, van den H.C.; Cordewener, J.H.G.; Nessen, M.A.; America, A.H.P.; Meer, van der I.M.

    2015-01-01

    Celiac disease (CD) is a food-related disease caused by certain gluten peptides containing T-cell stimulating epitopes from wheat, rye, and barley. CD-patients have to maintain a gluten-free diet and are therefore dependent on reliable testing and labeling of gluten-free products. So far, the

  3. Label-free detection of C-reactive protein using reflectometric interference spectroscopy-based sensing system

    Energy Technology Data Exchange (ETDEWEB)

    Choi, Hyung Woo; Sakata, Yasuhiko [Graduate School of Engineering, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe 657-8501 (Japan); Kurihara, Yoshikazu [Graduate School of Engineering, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe 657-8501 (Japan); KONICA MINOLTA OPTO, Inc., 1 Sakura-machi, Hino-shi, Tokyo 191-8511 (Japan); Ooya, Tooru [Graduate School of Engineering, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe 657-8501 (Japan); Takeuchi, Toshifumi, E-mail: takeuchi@gold.kobe-u.ac.jp [Graduate School of Engineering, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe 657-8501 (Japan)

    2012-05-30

    Highlights: Black-Right-Pointing-Pointer A new RIfS-based label-free biosensing system for C-reactive protein was developed. Black-Right-Pointing-Pointer Silicon-based inexpensive chips and the simple optical setup were employed. Black-Right-Pointing-Pointer Owing to the TMS treatment and the use of protein A, the sensitivity was enhanced. Black-Right-Pointing-Pointer It can be applied to other target as a substitute of SPR-based expensive sensors. - Abstract: Reflectometric interference spectroscopy (RIfS) is a label-free, time-resolved technique, and suitable for detecting antibody-antigen interaction. This work describes a continuous flow biosensor for C-reactive protein (CRP), involving an effective immobilization method of a monoclonal antibody against CRP (anti-CRP) to achieve highly sensitive RIfS-based detection of CRP. The silicon nitride-coated silicon chip (SiN chip) for the RIfS sensing was first treated with trimethylsilylchloride (TMS), followed by UV-light irradiation to in situ generation of homogeneous silanols on the surface. Following amination by 3-aminopropyltriethoxysilane, carboxymethyldextran (CMD) was grafted, and subsequently, protein A was immobilized to create the oriented anti-CRP surface. The immobilization process of protein A and anti-CRP was monitored with the RIfS system by consecutive injections of an amine coupling reagent, protein A and anti-CRP, respectively, to confirm the progress of each step in real time. The sensitivity was enhanced when all of the processes were adopted, suggesting that the oriented immobilization of anti-CRP via protein A that was coupled with the grafted CMD on the aminated surface of TMS-treated SiN chip. The feasibility of the present sensing system was demonstrated on the detection of CRP, where the silicon-based inexpensive chips and the simple optical setup were employed. It can be applied to other target molecules in various fields of life science as a substitute of surface plasmon resonance

  4. Surface stress sensor based on MEMS Fabry-Perot interferometer with high wavelength selectivity for label-free biosensing

    Science.gov (United States)

    Takahashi, Toshiaki; Hizawa, Takeshi; Misawa, Nobuo; Taki, Miki; Sawada, Kazuaki; Takahashi, Kazuhiro

    2018-05-01

    We have developed a surface stress sensor based on a microelectromechanical Fabry-Perot interferometer with high wavelength selectivity by using Au half-mirrors, for highly sensitive label-free biosensing. When the target molecule is adsorbed by the antigen-antibody reaction onto a movable membrane with a thin Au film, which acts as an upper mirror of the optical interferometer, the amount of deflection of the movable membrane deflected by the change in surface stress can be detected with high sensitivity. To improve the signal at the small membrane deflection region of this biosensor resulting in detection of low concentration molecules, by integrating 50 nm-thick Au half-mirrors, the wavelength selectivity of the optical interferometer has been successfully improved 6.6 times. Furthermore, the peak shift in the reflection spectrum due to the adsorption of bovine serum albumin (BSA) antigen with a concentration of 10 ng ml-l by the antigen-antibody reaction was spectroscopically measured on the fabricated optical interferometer, and the deflection amount of the movable membrane after 10 min treatment was 2.4 times larger than that of nonspecific adsorption with the avidin molecules. This result indicated that the proposed sensor can be used for selective detection of low-concentration target antigen molecules.

  5. Design Optimization of Structural Parameters for Highly Sensitive Photonic Crystal Label-Free Biosensors

    Directory of Open Access Journals (Sweden)

    Yun-ah Han

    2013-03-01

    Full Text Available The effects of structural design parameters on the performance of nano-replicated photonic crystal (PC label-free biosensors were examined by the analysis of simulated reflection spectra of PC structures. The grating pitch, duty, scaled grating height and scaled TiO2 layer thickness were selected as the design factors to optimize the PC structure. The peak wavelength value (PWV, full width at half maximum of the peak, figure of merit for the bulk and surface sensitivities, and surface/bulk sensitivity ratio were also selected as the responses to optimize the PC label-free biosensor performance. A parametric study showed that the grating pitch was the dominant factor for PWV, and that it had low interaction effects with other scaled design factors. Therefore, we can isolate the effect of grating pitch using scaled design factors. For the design of PC-label free biosensor, one should consider that: (1 the PWV can be measured by the reflection peak measurement instruments, (2 the grating pitch and duty can be manufactured using conventional lithography systems, and (3 the optimum design is less sensitive to the grating height and TiO2 layer thickness variations in the fabrication process. In this paper, we suggested a design guide for highly sensitive PC biosensor in which one select the grating pitch and duty based on the limitations of the lithography and measurement system, and conduct a multi objective optimization of the grating height and TiO2 layer thickness for maximizing performance and minimizing the influence of parameter variation. Through multi-objective optimization of a PC structure with a fixed grating height of 550 nm and a duty of 50%, we obtained a surface FOM of 66.18 RIU−1 and an S/B ratio of 34.8%, with a grating height of 117 nm and TiO2 height of 210 nm.

  6. Design optimization of structural parameters for highly sensitive photonic crystal label-free biosensors.

    Science.gov (United States)

    Ju, Jonghyun; Han, Yun-ah; Kim, Seok-min

    2013-03-07

    The effects of structural design parameters on the performance of nano-replicated photonic crystal (PC) label-free biosensors were examined by the analysis of simulated reflection spectra of PC structures. The grating pitch, duty, scaled grating height and scaled TiO2 layer thickness were selected as the design factors to optimize the PC structure. The peak wavelength value (PWV), full width at half maximum of the peak, figure of merit for the bulk and surface sensitivities, and surface/bulk sensitivity ratio were also selected as the responses to optimize the PC label-free biosensor performance. A parametric study showed that the grating pitch was the dominant factor for PWV, and that it had low interaction effects with other scaled design factors. Therefore, we can isolate the effect of grating pitch using scaled design factors. For the design of PC-label free biosensor, one should consider that: (1) the PWV can be measured by the reflection peak measurement instruments, (2) the grating pitch and duty can be manufactured using conventional lithography systems, and (3) the optimum design is less sensitive to the grating height and TiO2 layer thickness variations in the fabrication process. In this paper, we suggested a design guide for highly sensitive PC biosensor in which one select the grating pitch and duty based on the limitations of the lithography and measurement system, and conduct a multi objective optimization of the grating height and TiO2 layer thickness for maximizing performance and minimizing the influence of parameter variation. Through multi-objective optimization of a PC structure with a fixed grating height of 550 nm and a duty of 50%, we obtained a surface FOM of 66.18 RIU-1 and an S/B ratio of 34.8%, with a grating height of 117 nm and TiO2 height of 210 nm.

  7. Label-free detection of insulin and glucagon within human islets of Langerhans using Raman spectroscopy.

    Directory of Open Access Journals (Sweden)

    Janneke Hilderink

    Full Text Available Intrahepatic transplantation of donor islets of Langerhans is a promising therapy for patients with type 1 diabetes. It is of critical importance to accurately monitor islet quality before transplantation, which is currently done by standard histological methods that are performed off-line and require extensive sample preparation. As an alternative, we propose Raman spectroscopy which is a non-destructive and label-free technique that allows continuous real-time monitoring of the tissue to study biological changes as they occur. By performing Raman spectroscopic measurements on purified insulin and glucagon, we showed that the 520 cm(-1 band assigned to disulfide bridges in insulin, and the 1552 cm(-1 band assigned to tryptophan in glucagon are mutually exclusive and could therefore be used as indirect markers for the label-free distinction between both hormones. High-resolution hyperspectral Raman imaging for these bands showed the distribution of disulfide bridges and tryptophan at sub-micrometer scale, which correlated with the location of insulin and glucagon as revealed by conventional immunohistochemistry. As a measure for this correlation, quantitative analysis was performed comparing the Raman images with the fluorescence images, resulting in Dice coefficients (ranging between 0 and 1 of 0.36 for insulin and 0.19 for glucagon. Although the use of separate microscope systems with different spatial resolution and the use of indirect Raman markers cause some image mismatch, our findings indicate that Raman bands for disulfide bridges and tryptophan can be used as distinctive markers for the label-free detection of insulin and glucagon in human islets of Langerhans.

  8. Highly stable porous silicon-carbon composites as label-free optical biosensors.

    Science.gov (United States)

    Tsang, Chun Kwan; Kelly, Timothy L; Sailor, Michael J; Li, Yang Yang

    2012-12-21

    A stable, label-free optical biosensor based on a porous silicon-carbon (pSi-C) composite is demonstrated. The material is prepared by electrochemical anodization of crystalline Si in an HF-containing electrolyte to generate a porous Si template, followed by infiltration of poly(furfuryl) alcohol (PFA) and subsequent carbonization to generate the pSi-C composite as an optically smooth thin film. The pSi-C sensor is significantly more stable toward aqueous buffer solutions (pH 7.4 or 12) compared to thermally oxidized (in air, 800 °C), hydrosilylated (with undecylenic acid), or hydrocarbonized (with acetylene, 700 °C) porous Si samples prepared and tested under similar conditions. Aqueous stability of the pSi-C sensor is comparable to related optical biosensors based on porous TiO(2) or porous Al(2)O(3). Label-free optical interferometric biosensing with the pSi-C composite is demonstrated by detection of rabbit IgG on a protein-A-modified chip and confirmed with control experiments using chicken IgG (which shows no affinity for protein A). The pSi-C sensor binds significantly more of the protein A capture probe than porous TiO(2) or porous Al(2)O(3), and the sensitivity of the protein-A-modified pSi-C sensor to rabbit IgG is found to be ~2× greater than label-free optical biosensors constructed from these other two materials.

  9. Interface Layering Phenomena in Capacitance Detection of DNA with Biochips

    Directory of Open Access Journals (Sweden)

    Sandro Carrara

    2007-02-01

    Full Text Available Reliable DNA detection is of great importance for the development of the Lab-on-chip technology. The effort of the most recent projects on this field is to integrate all necessary operations, such as sample preparation (mixing, PCR amplification together with the sensor user for DNA detection. Among the different ways to sense the DNA hybridization, fluorescence based detection has been favored by the market. However, fluorescence based approaches require that the DNA targets are labeled by means of chromophores. As an alternative label-free DNA detection method, capacitance detection was recently proposed by different authors. While this effect has been successfully demonstrated by several groups, the model used for data analysis is far too simple to describe the real behavior of a DNA sensor. The aim of the present paper is to propose a different electrochemical model to describe DNA capacitance detection.

  10. Direct identification of amyloids by label-free quantitative LC-MS

    DEFF Research Database (Denmark)

    Dueholm, Morten Simonsen; Danielsen, Heidi Nolsøe; Hansen, Susan Hove

    adhesive and therefore bind to pipette tips and other consumables. Pure cultures, large sample volumes and high productivity of amyloids are therefore required for successful purification. We here present a quantitative proteomics technique that allow direct identification of functional amyloid candidates......Direct identification of amyloids by label-free quantitative LC-MS H. N. Danielsen, S. H. Hansen, F.-A. Herbst, P. H. Nielsen, M. S. Dueholm Amyloids are highly ordered fibrillar protein polymers used by organisms from all domains of life due to their exceptional properties. We have previously...... in complex samples based on their structural stability in the presence of increasing concentrations of formic acid....

  11. Label-Free Quantitation of Ribosomal Proteins from Bacillus subtilis for Antibiotic Research.

    Science.gov (United States)

    Schäkermann, Sina; Prochnow, Pascal; Bandow, Julia E

    2017-01-01

    Current research is focusing on ribosome heterogeneity as a response to changing environmental conditions and stresses, such as antibiotic stress. Altered stoichiometry and composition of ribosomal proteins as well as association of additional protein factors are mechanisms for shaping the protein expression profile or hibernating ribosomes. Here, we present a method for the isolation of ribosomes to analyze antibiotic-induced changes in the composition of ribosomes in Bacillus subtilis or other bacteria. Ribosomes and associated proteins are isolated by ultracentrifugation and proteins are identified and quantified using label-free mass spectrometry.

  12. A label free aptamer-based LPG sensor for detection of mercury in aquatic solutions

    Science.gov (United States)

    Nikbakht, Hamed; Latifi, Hamid; Ziaee, Farzaneh

    2015-09-01

    We demonstrate a label free fiber optic sensor for detection of mercury ions in aquatic solutions. This sensor utilizes aptamers as bio-recognition element which traps mercury ions and cause a refractive index change in the vicinity of the sensor. Refractive index variations lead to a change in the transmission spectrum that can be used to calculate the concentration of mercury ions in that solution. The concentration of 1 nM mercury ions was detected which is below the specific amount determined by the US environmental protection agency as the maximum authorized contaminant level of Hg2+ ions in drinking water.

  13. Comparison of serum fractionation methods by data independent label-free proteomics

    Directory of Open Access Journals (Sweden)

    D. Baiwir

    2015-12-01

    Full Text Available Off-line sample prefractionations applied prior to biomarker discovery proteomics are options to enable more protein identifications and detect low-abundance proteins. This work compared five commercial methods efficiency to raw serum analysis using label-free proteomics. The variability of the protein quantities determined for each process was similar to the unprefractionated serum. A 49% increase in protein identifications and 12.2% of reliable quantification were obtained. A 61 times lower limit of protein quantitation was reached compared to protein concentrations observed in raw serum. The concentrations of detected proteins were confronted to estimated reference values.

  14. Free-solution, label-free molecular interactions studied by back-scattering interferometry

    DEFF Research Database (Denmark)

    Bornhop, D.J.; Latham, J.C.; Kussrow, A.

    2007-01-01

    Free-solution, label-free molecular interactions were investigated with back-scattering interferometry in a simple optical train composed of a helium-neon laser, a microfluidic channel, and a position sensor. Molecular binding interactions between proteins, ions and protein, and small molecules...... and protein, were determined with high dynamic range dissociation constants (K-d spanning six decades) and unmatched sensitivity (picomolar K-d's and detection limits of 10,000s of molecules). With this technique, equilibrium dissociation constants were quantified for protein A and immunoglobulin G...

  15. Label-free visualization of ultrastructural features of artificial synapses via cryo-EM.

    Science.gov (United States)

    Gopalakrishnan, Gopakumar; Yam, Patricia T; Madwar, Carolin; Bostina, Mihnea; Rouiller, Isabelle; Colman, David R; Lennox, R Bruce

    2011-12-21

    The ultrastructural details of presynapses formed between artificial substrates of submicrometer silica beads and hippocampal neurons are visualized via cryo-electron microscopy (cryo-EM). The silica beads are derivatized by poly-d-lysine or lipid bilayers. Molecular features known to exist at presynapses are clearly present at these artificial synapses, as visualized by cryo-EM. Key synaptic features such as the membrane contact area at synaptic junctions, the presynaptic bouton containing presynaptic vesicles, as well as microtubular structures can be identified. This is the first report of the direct, label-free observation of ultrastructural details of artificial synapses.

  16. Quantitative Label-Free Cell Proliferation Tracking with a Versatile Electrochemical Impedance Detection Platform

    DEFF Research Database (Denmark)

    Caviglia, Claudia; Carminati, M; Heiskanen, Arto

    2012-01-01

    optimal detection strategies. Electrochemical Impedance Spectroscopy (EIS) has been used to monitor and compare adhesion of different cell lines. HeLa cells and 3T3 fibroblasts have been cultured for 12 hours on interdigitated electrode arrays integrated into a tailor-made cell culture platform. Both......Since the use of impedance measurements for label-free monitoring of cells has become widespread but still the choice of sensing configuration is not unique though crucial for a quantitative interpretation of data, we demonstrate the application of a novel custom multipotentiostat platform to study...... vertical and coplanar interdigitated sensing configuration approaches have been used and compared on the same cell populations....

  17. Organic liquids-responsive β-cyclodextrin-functionalized graphene-based fluorescence probe: label-free selective detection of tetrahydrofuran.

    Science.gov (United States)

    Hu, Huawen; Xin, John H; Hu, Hong; Wang, Xiaowen; Lu, Xinkun

    2014-06-06

    In this study, a label-free graphene-based fluorescence probe used for detection of volatile organic liquids was fabricated by a simple, efficient and low-cost method. To fabricate the probe, a bio-based β-cyclodextrin (β-CD) was firstly grafted on reduced graphene surfaces effectively and uniformly, as evidenced by various characterization techniques such as Ultraviolet/Visible spectroscopy, Fourier transform infrared spectroscopy, X-ray diffraction, thermogravimetric analysis, scanning electron microscopy and transmission electron microscopy. The subsequent inclusion of Rhodamine B (RhB) into the inner cavities of the β-CD grafted on the graphene surfaces was achieved easily by a solution mixing method, which yielded the graphene-based fluorescent switch-on probe. In addition, the gradual and controllable quenching of RhB by Fluorescence Resonance Energy Transfer from RhB to graphene during the process of stepwise accommodation of the RhB molecules into the β-CD-functionalized graphene was investigated in depth. A wide range of organic solvents was examined using the as-fabricated fluorescence probe, which revealed the highest sensitivity to tetrahydrofuran with the detection limit of about 1.7 μg/mL. Some insight into the mechanism of the different responsive behaviors of the fluorescence sensor to the examined targets was also described.

  18. Label-free and direct detection of C-reactive protein using reduced graphene oxide-nanoparticle hybrid impedimetric sensor.

    Science.gov (United States)

    Yagati, Ajay Kumar; Pyun, Jae-Chul; Min, Junhong; Cho, Sungbo

    2016-02-01

    For label-free and direct detection of C-reactive protein (CRP), an impedimetric sensor based on an indium tin oxide (ITO) electrode array functionalized with reduced graphene oxide-nanoparticle (rGO-NP) hybrid was fabricated and evaluated. Analytical measurements were performed to examine the properties of rGO-NP-modified ITO microelectrodes and to determine the influence upon sensory performance of using nanostructures modified for antibody immobilization and for recognition of CRP binding events. Impedimetric measurements in the presence of the redox couple [Fe(CN)6](3-/4-) showed significant changes in charge transfer resistance upon binding of CRP. The impedance measurements were highly target specific, linear with logarithmic CRP concentrations in PBS and human serum across a 1 ng mL(-1) and 1000 ng mL(-1) range and associated with a detection limits of 0.06 and 0.08 ng mL(-1) respectively. Copyright © 2015 Elsevier B.V. All rights reserved.

  19. Fast label-free detection of Legionella spp. in biofilms by applying immunomagnetic beads and Raman spectroscopy.

    Science.gov (United States)

    Kusić, Dragana; Rösch, Petra; Popp, Jürgen

    2016-03-01

    Legionellae colonize biofilms, can form a biofilm by itself and multiply intracellularly within the protozoa commonly found in water distribution systems. Approximately half of the known species are pathogenic and have been connected to severe multisystem Legionnaires' disease. The detection methods for Legionella spp. in water samples are still based on cultivation, which is time consuming due to the slow growth of this bacterium. Here, we developed a cultivation-independent, label-free and fast detection method for legionellae in a biofilm matrix based on the Raman spectroscopic analysis of isolated single cells via immunomagnetic separation (IMS). A database comprising the Raman spectra of single bacterial cells captured and separated from the biofilms formed by each species was used to build the identification method based on a support vector machine (SVM) discriminative classifier. The complete method allows the detection of Legionella spp. in 100 min. Cross-reactivity of Legionella spp. specific immunomagnetic beads to the other studied genera was tested, where only small cell amounts of Pseudomonas aeruginosa, Klebsiella pneumoniae and Escherichia coli compared to the initial number of cells were isolated by the immunobeads. Nevertheless, the Raman spectra collected from isolated non-targeted bacteria were well-discriminated from the Raman spectra collected from isolated Legionella cells, whereby the Raman spectra of the independent dataset of Legionella strains were assigned with an accuracy of 98.6%. In addition, Raman spectroscopy was also used to differentiate between isolated Legionella species. Copyright © 2016 Elsevier GmbH. All rights reserved.

  20. Organic Liquids-Responsive β-Cyclodextrin-Functionalized Graphene-Based Fluorescence Probe: Label-Free Selective Detection of Tetrahydrofuran

    Directory of Open Access Journals (Sweden)

    Huawen Hu

    2014-06-01

    Full Text Available In this study, a label-free graphene-based fluorescence probe used for detection of volatile organic liquids was fabricated by a simple, efficient and low-cost method. To fabricate the probe, a bio-based β-cyclodextrin (β-CD was firstly grafted on reduced graphene surfaces effectively and uniformly, as evidenced by various characterization techniques such as Ultraviolet/Visible spectroscopy, Fourier transform infrared spectroscopy, X-ray diffraction, thermogravimetric analysis, scanning electron microscopy and transmission electron microscopy. The subsequent inclusion of Rhodamine B (RhB into the inner cavities of the β-CD grafted on the graphene surfaces was achieved easily by a solution mixing method, which yielded the graphene-based fluorescent switch-on probe. In addition, the gradual and controllable quenching of RhB by Fluorescence Resonance Energy Transfer from RhB to graphene during the process of stepwise accommodation of the RhB molecules into the β-CD-functionalized graphene was investigated in depth. A wide range of organic solvents was examined using the as-fabricated fluorescence probe, which revealed the highest sensitivity to tetrahydrofuran with the detection limit of about 1.7 μg/mL. Some insight into the mechanism of the different responsive behaviors of the fluorescence sensor to the examined targets was also described.

  1. Label-free in situ imaging of oil body dynamics and chemistry in germination.

    Science.gov (United States)

    Waschatko, Gustav; Billecke, Nils; Schwendy, Sascha; Jaurich, Henriette; Bonn, Mischa; Vilgis, Thomas A; Parekh, Sapun H

    2016-10-01

    Plant oleosomes are uniquely emulsified lipid reservoirs that serve as the primary energy source during seed germination. These oil bodies undergo significant changes regarding their size, composition and structure during normal seedling development; however, a detailed characterization of these oil body dynamics, which critically affect oil body extractability and nutritional value, has remained challenging because of a limited ability to monitor oil body location and composition during germination in situ Here, we demonstrate via in situ, label-free imaging that oil bodies are highly dynamic intracellular organelles that are morphologically and biochemically remodelled extensively during germination. Label-free, coherent Raman microscopy (CRM) combined with bulk biochemical measurements revealed the temporal and spatial regulation of oil bodies in native soya bean cotyledons during the first eight days of germination. Oil bodies undergo a cycle of growth and shrinkage that is paralleled by lipid and protein compositional changes. Specifically, the total protein concentration associated with oil bodies increases in the first phase of germination and subsequently decreases. Lipids contained within the oil bodies change in saturation and chain length during germination. Our results show that CRM is a well-suited platform to monitor in situ lipid dynamics and local chemistry and that oil bodies are actively remodelled during germination. This underscores the dynamic role of lipid reservoirs in plant development. © 2016 The Authors.

  2. Label-free amino acid detection based on nanocomposites of graphene oxide hybridized with gold nanoparticles.

    Science.gov (United States)

    Zhang, Qian; Zhang, Diming; Lu, Yanli; Xu, Gang; Yao, Yao; Li, Shuang; Liu, Qingjun

    2016-03-15

    Nanocomposites of graphene oxide and gold nanoparticles (GO/GNPs) were synthesized for label-free detections of amino acids. Interactions between the composites and amino acids were investigated by both naked-eye observation and optical absorption spectroscopy. The GO/GNPs composites displayed apparent color changes and absorption spectra changes in presences of amino acids including glutamate, aspartate, and cysteine. The interaction mechanisms of the composites and amino acids were discussed and explored with sulfhydryl groups and non-α-carboxylic groups on the amino acids. Sensing properties of the composites were tested, while pure gold particles were used as the control. The results suggested that the GO/GNPs composites had better linearity and stability in dose-dependent responses to the amino acids than those of the particles, especially in detections for acidic amino acids. Therefore, the nanocomposites platform can provide a convenient and efficient approach for label-free optical detections of important molecules such as amino acids. Copyright © 2015 Elsevier B.V. All rights reserved.

  3. Compact 3D printed module for fluorescence and label-free imaging using evanescent excitation

    Science.gov (United States)

    Pandey, Vikas; Gupta, Shalini; Elangovan, Ravikrishnan

    2018-01-01

    Total internal reflection fluorescence (TIRF) microscopy is widely used for selective excitation and high-resolution imaging of fluorophores, and more recently label-free nanosized objects, with high vertical confinement near a liquid-solid interface. Traditionally, high numerical aperture objectives (>1.4) are used to simultaneously generate evanescent waves and collect fluorescence emission signals which limits their use to small area imaging (filters to prevent specular reflection within the objective lenses. We have developed a compact 3D module called cTIRF that can generate evanescent waves in microscope glass slides via a planar waveguide illumination. The module can be attached as a fixture to any existing optical microscope, converting it into a TIRF and enabling high signal-to-noise ratio (SNR) fluorescence imaging using any magnification objective. As the incidence optics is perpendicular to the detector, label-free evanescent scattering-based imaging of submicron objects can also be performed without using emission filters. SNR is significantly enhanced in this case as compared to cTIRF alone, as seen through our model experiments performed on latex beads and mammalian cells. Extreme flexibility and the low cost of our approach makes it scalable for limited resource settings.

  4. A compact and portable optofluidic device for detection of liquid properties and label-free sensing

    Science.gov (United States)

    Lahoz, F.; Martín, I. R.; Walo, D.; Gil-Rostra, J.; Yubero, F.; Gonzalez-Elipe, A. R.

    2017-06-01

    Optofluidic lasers have been widely investigated over the last few years mainly because they can be easily integrated in sensor devices. However, high power pulse lasers are required as excitation sources, which, in practice, limit the portability of the system. Trying to overcome some of these limitations, in this paper we propose the combined use of a small CW laser with a Fabry-Perot optofluidic planar microcavity showing high sensitivity and versatility for detection of liquid properties and label-free sensing. Firstly, a fluorescein solution in ethanol is used to demonstrate the high performances of the FP microcavity as a temperature sensor both in the laser (high pump power above laser threshold) and in the fluorescence (low pump power) regimes. A shift in the wavelength of the resonant cavity modes is used to detect changes in the temperature and our results show that high sensitivities could be already obtained using cheap and portable CW diode lasers. In the second part of the paper, the demonstration of this portable device for label-free sensing is illustrated under low CW pumping. The wavelength positions of the optofluidic resonant modes are used to detect glucose concentrations in water solutions using a protein labelled with a fluorescent dye as the active medium.

  5. 3D label-free prostate specific antigen (PSA) immunosensor based on graphene-gold composites.

    Science.gov (United States)

    Jang, Hee Dong; Kim, Sun Kyung; Chang, Hankwon; Choi, Jeong-Woo

    2015-01-15

    Highly sensitive and label-free detection of the prostate specific antigen (PSA) remains a challenge in the diagnosis of prostate cancer. Here, a novel three-dimensional (3D) electrochemical immunosensor capable of sensitive and label-free detection of PSA is reported. This unique immunosensor is equipped with a highly conductive graphene (GR)-based gold (Au) composite modified electrode. The GR-based Au composite is prepared using aerosol spray pyrolysis and the morphology of the composite is the shape of a crumpled GR ball decorated with Au nanoparticles. Unlike the previous research, this novel 3D immunosensor functions very well over a broad linear range of 0-10 ng/mL with a low detection limit of 0.59 ng/mL; furthermore, it exhibits a significantly increased electron transfer and high sensitivity toward PSA. The highest rate of current change with respect to the PSA concentration is 5 μA/(ng/mL). Satisfactory selectivity, reproducibility, and stability of the 3D immunosensor are also exhibited. Copyright © 2014 Elsevier B.V. All rights reserved.

  6. Nanogap biosensors for electrical and label-free detection of biomolecular interactions

    International Nuclear Information System (INIS)

    Kyu Kim, Sang; Cho, Hyunmin; Park, Hye-Jung; Kwon, Dohyoung; Min Lee, Jeong; Hyun Chung, Bong

    2009-01-01

    We demonstrate nanogap biosensors for electrical and label-free detection of biomolecular interactions. Parallel fabrication of nanometer distance gaps has been achieved using a silicon anisotropic wet etching technique on a silicon-on-insulator (SOI) wafer with a finely controllable silicon device layer. Since silicon anisotropic wet etching resulted in a trapezoid-shaped structure whose end became narrower during the etching, the nanogap structure was simply fabricated on the device layer of a SOI wafer. The nanogap devices were individually addressable and a gap size of less than 60 nm was obtained. We demonstrate that the nanogap biosensors can electrically detect biomolecular interactions such as biotin/streptavidin and antigen/antibody pairs. The nanogap devices show a current increase when the proteins are bound to the surface. The current increases proportionally depending upon the concentrations of the molecules in the range of 100 fg ml -1 -100 ng ml -1 at 1 V bias. It is expected that the nanogap developed here could be a highly sensitive biosensor platform for label-free detection of biomolecular interactions.

  7. Ultrasensitive Sensing Material Based on Opal Photonic Crystal for Label-Free Monitoring of Transferrin.

    Science.gov (United States)

    Wu, Enqi; Peng, Yuan; Zhang, Xihao; Bai, Jialei; Song, Yanqiu; He, Houluo; Fan, Longxing; Qu, Xiaochen; Gao, Zhixian; Liu, Ying; Ning, Baoan

    2017-02-22

    A new opal photonic crystal (PC) sensing material, allowing label-free detection of transferrin (TRF), is proposed in the current study. This photonic crystal was prepared via a vertical convective self-assembly method with monodisperse microspheres polymerized by methyl methacrylate (MMA) and 3-acrylamidophenylboronic acid (AAPBA). FTIR, TG, and DLS were used to characterize the components and particle size of the monodisperse microspheres. SEM was used to observe the morphology of the PC. The diffraction peak intensity decreases as the TRF concentration increase. This was due to the combination of TRF to the boronic acid group of the photonic crystal. After condition optimization, a standard curve was obtained and the linear range of TRF concentration was from 2 × 10 -3 ng/mL to 200 ng/mL. Measurement of TRF concentration in simulated urine sample was also investigated using the sensing material. The results indicated that the PC provided a cheap, label-free, and easy-to-use alternative for TRF determination in clinical diagnostics.

  8. Probing Xylan-Specific Raman Bands for Label-Free Imaging Xylan in Plant Cell Wall

    Energy Technology Data Exchange (ETDEWEB)

    Zeng, Yining; Yarbrough, John M.; Mittal, Ashutosh; Tucker, Melvin P.; Vinzant, Todd; Himmel, Michael E.

    2015-06-15

    Xylan constitutes a significant portion of biomass (e.g. 22% in corn stover used in this study). Xylan is also an important source of carbohydrates, besides cellulose, for renewable and sustainable energy applications. Currently used method for the localization of xylan in biomass is to use fluorescence confocal microscope to image the fluorescent dye labeled monoclonal antibody that specifically binds to xylan. With the rapid adoption of the Raman-based label-free chemical imaging techniques in biology, identifying Raman bands that are unique to xylan would be critical for the implementation of the above label-free techniques for in situ xylan imaging. Unlike lignin and cellulose that have long be assigned fingerprint Raman bands, specific Raman bands for xylan remain unclear. The major challenge is the cellulose in plant cell wall, which has chemical units highly similar to that of xylan. Here we report using xylanase to specifically remove xylan from feedstock. Under various degree of xylan removal, with minimum impact to other major cell wall components, i.e. lignin and cellulose, we have identified Raman bands that could be further tested for chemical imaging of xylan in biomass in situ.

  9. A Label Free Disposable Device for Rapid Isolation of Rare Tumor Cells from Blood by Ultrasounds

    Directory of Open Access Journals (Sweden)

    Itziar González

    2018-03-01

    Full Text Available The use of blood samples as liquid biopsy is a label-free method for cancer diagnosis that offers benefits over traditional invasive biopsy techniques. Cell sorting by acoustic waves offers a means to separate rare cells from blood samples based on their physical properties in a label-free, contactless and biocompatible manner. Herein, we describe a flow-through separation approach that provides an efficient separation of tumor cells (TCs from white blood cells (WBCs in a microfluidic device, “THINUS-Chip” (Thin-Ultrasonic-Separator-Chip, actuated by ultrasounds. We introduce for the first time the concept of plate acoustic waves (PAW applied to acoustophoresis as a new strategy. It lies in the geometrical chip design: different to other microseparators based on either bulk acoustic waves (BAW or surface waves (SAW, SSAW and tSAW, it allows the use of polymeric materials without restrictions in the frequency of work. We demonstrate its ability to perform high-throughput isolation of TCs from WBCs, allowing a recovery rate of 84% ± 8% of TCs with a purity higher than 80% and combined viability of 85% at a flow rate of 80 μL/min (4.8 mL/h. The THINUS-Chip performs cell fractionation with low-cost manufacturing processes, opening the door to possible easy printing fabrication.

  10. Label-free nonlinear optical microscopy detects early markers for osteogenic differentiation of human stem cells

    Science.gov (United States)

    Hofemeier, Arne D.; Hachmeister, Henning; Pilger, Christian; Schürmann, Matthias; Greiner, Johannes F. W.; Nolte, Lena; Sudhoff, Holger; Kaltschmidt, Christian; Huser, Thomas; Kaltschmidt, Barbara

    2016-05-01

    Tissue engineering by stem cell differentiation is a novel treatment option for bone regeneration. Most approaches for the detection of osteogenic differentiation are invasive or destructive and not compatible with live cell analysis. Here, non-destructive and label-free approaches of Raman spectroscopy, coherent anti-Stokes Raman scattering (CARS) and second harmonic generation (SHG) microscopy were used to detect and image osteogenic differentiation of human neural crest-derived inferior turbinate stem cells (ITSCs). Combined CARS and SHG microscopy was able to detect markers of osteogenesis within 14 days after osteogenic induction. This process increased during continued differentiation. Furthermore, Raman spectroscopy showed significant increases of the PO43- symmetric stretch vibrations at 959 cm-1 assigned to calcium hydroxyapatite between days 14 and 21. Additionally, CARS microscopy was able to image calcium hydroxyapatite deposits within 14 days following osteogenic induction, which was confirmed by Alizarin Red-Staining and RT- PCR. Taken together, the multimodal label-free analysis methods Raman spectroscopy, CARS and SHG microscopy can monitor osteogenic differentiation of adult human stem cells into osteoblasts with high sensitivity and spatial resolution in three dimensions. Our findings suggest a great potential of these optical detection methods for clinical applications including in vivo observation of bone tissue-implant-interfaces or disease diagnosis.

  11. Label-free protein profiling of formalin-fixed paraffin-embedded (FFPE) heart tissue reveals immediate mitochondrial impairment after ionising radiation.

    Science.gov (United States)

    Azimzadeh, Omid; Scherthan, Harry; Yentrapalli, Ramesh; Barjaktarovic, Zarko; Ueffing, Marius; Conrad, Marcus; Neff, Frauke; Calzada-Wack, Julia; Aubele, Michaela; Buske, Christian; Atkinson, Michael J; Hauck, Stefanie M; Tapio, Soile

    2012-04-18

    Qualitative proteome profiling of formalin-fixed, paraffin-embedded (FFPE) tissue is advancing the field of clinical proteomics. However, quantitative proteome analysis of FFPE tissue is hampered by the lack of an efficient labelling method. The usage of conventional protein labelling on FFPE tissue has turned out to be inefficient. Classical labelling targets lysine residues that are blocked by the formalin treatment. The aim of this study was to establish a quantitative proteomics analysis of FFPE tissue by combining the label-free approach with optimised protein extraction and separation conditions. As a model system we used FFPE heart tissue of control and exposed C57BL/6 mice after total body irradiation using a gamma ray dose of 3 gray. We identified 32 deregulated proteins (p≤0.05) in irradiated hearts 24h after the exposure. The proteomics data were further evaluated and validated by bioinformatics and immunoblotting investigation. In good agreement with our previous results using fresh-frozen tissue, the analysis indicated radiation-induced alterations in three main biological pathways: respiratory chain, lipid metabolism and pyruvate metabolism. The label-free approach enables the quantitative measurement of radiation-induced alterations in FFPE tissue and facilitates retrospective biomarker identification using clinical archives. Copyright © 2012 Elsevier B.V. All rights reserved.

  12. Label-free detection of gliadin food allergen mediated by real-time apta-PCR.

    Science.gov (United States)

    Pinto, Alessandro; Polo, Pedro Nadal; Henry, Olivier; Redondo, M Carmen Bermudo; Svobodova, Marketa; O'Sullivan, Ciara K

    2014-01-01

    Celiac disease is an immune-mediated enteropathy triggered by the ingestion of gluten. The only effective treatment consists in a lifelong gluten-free diet, requiring the food industry to tightly control the gluten contents of their products. To date, several gluten quantification approaches using antibodies are available and recommended by the legal authorities, such as Codex Alimentarius. However, whilst these antibody-based tests exhibit high sensitivity and specificity, the production of antibodies inherently requires the killing of host animals and is time-consuming and relatively expensive. Aptamers are structured single-stranded nucleic acid ligands that bind with high affinity and specificity to their cognate target, and aiming for a cost-effective viable alternative to the use of antibodies. Herein, we report the systematic evolution of ligands by exponential enrichment (SELEX)-based selection of a DNA aptamer against gliadin from a combinatorial DNA library and its application in a novel detection assay. Taking into account the hydrophobic nature of the gliadin target, a microtitre plate format was exploited for SELEX, where the target was immobilised via hydrophobic interactions, thus exposing aptatopes accessible for interaction with the DNA library. Evolution was followed using surface plasmon resonance, and following eight rounds of SELEX, the enriched DNA pool was cloned, sequenced and a clear consensus motif was identified. An apta-PCR assay was developed where competition for the aptamer takes place between the surface-immobilised gliadin and gliadin in the target sample, akin to an ELISA competitive format where the more target present in the sample, the less aptamer will bind to the immobilised gliadin. Following competition, any aptamer bound to the immobilised gliadin was heat-eluted and quantitatively amplified using real-time PCR, achieving a detection limit of approx. 2 nM (100 ng mL(-1)). The specificity of the selected aptamer was

  13. Immunogenicity of DNA vaccines encoding simian immunodeficiency virus antigen targeted to dendritic cells in rhesus macaques.

    Directory of Open Access Journals (Sweden)

    Matthias Tenbusch

    Full Text Available BACKGROUND: Targeting antigens encoded by DNA vaccines to dendritic cells (DCs in the presence of adjuvants enhances their immunogenicity and efficacy in mice. METHODOLOGY/PRINCIPAL FINDINGS: To explore the immunogenicity of this approach in non-human primates, we generated a single chain antibody to the antigen uptake receptor DEC-205 expressed on rhesus macaque DCs. DNA vaccines encoding this single chain antibody fused to the SIV capsid protein were delivered to six monkeys each by either intramuscular electroporation or conventional intramuscular injection co-injected or not with poly ICLC, a stabilized poly I: C analogue, as adjuvant. Antibodies to capsid were induced by the DC-targeting and non-targeting control DNA delivered by electroporation while conventional DNA immunization at a 10-fold higher dose of DNA failed to induce detectable humoral immune responses. Substantial cellular immune responses were also observed after DNA electroporation of both DNAs, but stronger responses were induced by the non-targeting vaccine. Conventional immunization with the DC-targeting DNA at a 10-fold higher dose did not give rise to substantial cellular immune responses, neither when co-injected with poly ICLC. CONCLUSIONS/SIGNIFICANCE: The study confirms the potent immunogenicity of DNA vaccines delivered by electroporation. Targeting the DNA via a single chain antibody to DEC-205 expressed by DCs, however, does not improve the immunogenicity of the antigens in non-human primates.

  14. Carbon nanostructure-based field-effect transistors for label-free chemical/biological sensors.

    Science.gov (United States)

    Hu, PingAn; Zhang, Jia; Li, Le; Wang, Zhenlong; O'Neill, William; Estrela, Pedro

    2010-01-01

    Over the past decade, electrical detection of chemical and biological species using novel nanostructure-based devices has attracted significant attention for chemical, genomics, biomedical diagnostics, and drug discovery applications. The use of nanostructured devices in chemical/biological sensors in place of conventional sensing technologies has advantages of high sensitivity, low decreased energy consumption and potentially highly miniaturized integration. Owing to their particular structure, excellent electrical properties and high chemical stability, carbon nanotube and graphene based electrical devices have been widely developed for high performance label-free chemical/biological sensors. Here, we review the latest developments of carbon nanostructure-based transistor sensors in ultrasensitive detection of chemical/biological entities, such as poisonous gases, nucleic acids, proteins and cells.

  15. Direct growth of graphene on quartz substrates for label-free detection of adenosine triphosphate.

    Science.gov (United States)

    Xu, Shicai; Man, Baoyuan; Jiang, Shouzhen; Yue, Weiwei; Yang, Cheng; Liu, Mei; Chen, Chuansong; Zhang, Chao

    2014-04-25

    We demonstrate that continuous, uniform graphene films can be directly synthesized on quartz substrates using a two-temperature-zone chemical vapor deposition system and that their layers can be controlled by adjusting the precursor partial pressure. Raman spectroscopy and transmission electron microscopy confirm the formation of monolayer graphene with a grain size of ∼100 nm. Hall measurements show a room-temperature carrier mobility above 1500 cm2 V(-1) s(-1). The optical transmittance and conductance of the graphene films are comparable to those of transferred metal-catalyzed graphene. The method avoids the complicated and skilled post-growth transfer process and allows the graphene to be directly incorporated into a fully functional biosensor for label-free detection of adenosine triphosphate (ATP). This device shows a fast response time of a few milliseconds and achieves a high sensitivity to ATP molecules over a very wide range from 0.002 to 5 mM.

  16. Label-free identification of intestinal metaplasia in the stomach using multiphoton microscopy

    International Nuclear Information System (INIS)

    Wu, G; Wei, J; Zheng, Z; Ye, J; Zeng, S

    2014-01-01

    The early diagnosis of intestinal metaplasia (IM) in the stomach together with effective therapeutic interventions is crucial to reducing the mortality-rates of the patients associated with gastric cancer. However, it is challenging during conventional white-light endoscopy, and histological analysis remains the ‘gold standard’ for the final diagnosis. Here, we describe a label-free imaging method, multiphoton microscopy (MPM), for the identification of IM in the stomach. It was found that multiphoton imaging provides cellular and subcellular details to the identification of IM from normal gastric tissues. In particular, there is significant difference in the population density of goblet cells between normal and IM gastric tissues, providing substantial potential to become a quantitative intrinsic marker for in vivo clinical diagnosis of early gastric lesions. To our knowledge, this is the first demonstration of the potential of MPM for the identification of IM. (letters)

  17. Asymmetric split-ring resonator-based biosensor for detection of label-free stress biomarkers

    Science.gov (United States)

    Lee, Hee-Jo; Lee, Jung-Hyun; Choi, Suji; Jang, Ik-Soon; Choi, Jong-Soon; Jung, Hyo-Il

    2013-07-01

    In this paper, an asymmetric split-ring resonator, metamaterial element, is presented as a biosensing transducer for detection of highly sensitive and label-free stress biomarkers. In particular, the two biomarkers, cortisol and α-amylase, are used for evaluating the sensitivity of the proposed biosensor. In case of cortisol detection, the competitive reaction between cortisol-bovine serum albumin and free cortisol is employed, while alpha-amylase is directly detected by its antigen-antibody reaction. From the experimental results, we find that the limit of detection and sensitivity of the proposed sensing device are about 1 ng/ml and 1.155 MHz/ng ml-1, respectively.

  18. Improvements in low-cost label-free QPI microscope for live cell imaging

    Science.gov (United States)

    Seniya, C.; Towers, C. E.; Towers, D. P.

    2017-07-01

    This paper reports an improvement in the development of a low-cost QPI microscope offering new capabilities in term of phase measurement accuracy for label-free live samples in the longer term (i.e., hours to days). The spatially separated scattered and non-scattered image light fields are reshaped in the Fourier plane and modulated to form an interference image at a CCD camera. The apertures that enable these two beams to be generated have been optimised by means of laser-cut apertures placed on the mirrors of a Michelson interferometer and has improved the phase measuring and reconstruction capability of the QPI microscope. The microscope was tested with transparent onion cells as an object of interest.

  19. Portable microfluidic raman system for rapid, label-free early disease signature detection

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Meiye [Sandia National Laboratories (SNL-CA), Livermore, CA (United States); Davis, Ryan Wesley [Sandia National Laboratories (SNL-CA), Livermore, CA (United States); Hatch, Anson [Sandia National Laboratories (SNL-CA), Livermore, CA (United States)

    2015-09-01

    In the early stages of infection, patients develop non-specific or no symptoms at all. While waiting for identification of the infectious agent, precious window of opportunity for early intervention is lost. The standard diagnostics require affinity reagents and sufficient pathogen titers to reach the limit of detection. In the event of a disease outbreak, triaging the at-risk population rapidly and reliably for quarantine and countermeasure is more important than the identification of the pathogen by name. To expand Sandia's portfolio of Biological threat management capabilities, we will utilize Raman spectrometry to analyze immune subsets in whole blood to rapidly distinguish infected from non-infected, and bacterial from viral infection, for the purpose of triage during an emergency outbreak. The goal of this one year LDRD is to determine whether Raman spectroscopy can provide label-free detection of early disease signatures, and define a miniaturized Raman detection system meeting requirements for low- resource settings.

  20. Design of a Label-Free, Distributed Bragg Grating Resonator Based Dielectric Waveguide Biosensor

    Directory of Open Access Journals (Sweden)

    Florian Kehl

    2015-01-01

    Full Text Available In this work, we present a resonant, dielectric waveguide device based on distributed Bragg gratings for label-free biosensing applications. The refractive index sensitive optical transducer aims at improving the performance of planar waveguide grating sensor systems with limited Q-factor and dynamic range by combing the advantages of resonant cavities, such as a multitude of resonance peaks with high finesse, with the manageable complexity of waveguide grating couplers. The general sensor concept is introduced and supported by theoretical considerations as well as numerical simulations based on Coupled Mode Theory. In contrast to a single Bragg grating reflector, the presented Fabry-Pérot type distributed Bragg resonator exhibits an extended measurement range as well as relaxed fabrication tolerances. The resulting, relatively simple sensor structure can be fabricated with standard lithographic means and is independent of expensive light-sources and/or detectors, making an affordable but sensitive device, potentially suitable for point-of-care applications.

  1. Nanosensors for label-free measurement of sodium ion fluxes of neuronal cells

    Energy Technology Data Exchange (ETDEWEB)

    Gebinoga, Michael, E-mail: michael.gebinoga@tu-ilmenau.de [ZIK MacroNano Microfluidics and Biosensors, Technical University Ilmenau, P.O. Box 100565, D-98684 Ilmenau (Germany); Silveira, Liele; Cimalla, Irina [ZIK MacroNano Microfluidics and Biosensors, Technical University Ilmenau, P.O. Box 100565, D-98684 Ilmenau (Germany); Dumitrescu, Andreea [University of Pennsylvania - School of Engineering and Applied Science, Philadelphia, PA 19104-6391 (United States); Kittler, Mario; Luebbers, Benedikt; Becker, Annette [ZIK MacroNano Microfluidics and Biosensors, Technical University Ilmenau, P.O. Box 100565, D-98684 Ilmenau (Germany); Lebedev, Vadim [Fraunhofer Institute for Solid State Physics, Tullastr. 7, D-79108 Freiburg (Germany); Schober, Andreas [ZIK MacroNano Microfluidics and Biosensors, Technical University Ilmenau, P.O. Box 100565, D-98684 Ilmenau (Germany)

    2010-05-25

    Novel nanosensors based on aluminium gallium nitrides (AlGaN/GaN) high electron mobility transistors have been of high interest during the last years, especially for their electrical characteristics as open gate field effect transistors. These nanosensors provide a valuable tool for high content screening in drug discovery, cell monitoring and liquid analyses focusing on applications of electrochemical detection technology. Our own measurements with these sensors confirm their pH sensitivity and in addition the possibility of detection of other ions in aqueous media. These measurements deal with the reactions of NG 108-15 (mouse neuroblastoma x rat glioma hybrid) neuronal cells in response to different acetylcholinesterase inhibitors. Our experimental approach shows some advantages. The first advantage is the label-free measurement of ion fluxes, and another advantage is the possibility non-destructively to estimate cell signals.

  2. Nanosensors for label-free measurement of sodium ion fluxes of neuronal cells

    International Nuclear Information System (INIS)

    Gebinoga, Michael; Silveira, Liele; Cimalla, Irina; Dumitrescu, Andreea; Kittler, Mario; Luebbers, Benedikt; Becker, Annette; Lebedev, Vadim; Schober, Andreas

    2010-01-01

    Novel nanosensors based on aluminium gallium nitrides (AlGaN/GaN) high electron mobility transistors have been of high interest during the last years, especially for their electrical characteristics as open gate field effect transistors. These nanosensors provide a valuable tool for high content screening in drug discovery, cell monitoring and liquid analyses focusing on applications of electrochemical detection technology. Our own measurements with these sensors confirm their pH sensitivity and in addition the possibility of detection of other ions in aqueous media. These measurements deal with the reactions of NG 108-15 (mouse neuroblastoma x rat glioma hybrid) neuronal cells in response to different acetylcholinesterase inhibitors. Our experimental approach shows some advantages. The first advantage is the label-free measurement of ion fluxes, and another advantage is the possibility non-destructively to estimate cell signals.

  3. Label-Free Imaging and Biochemical Characterization of Bovine Sperm Cells

    Science.gov (United States)

    Ferrara, Maria Antonietta; Di Caprio, Giuseppe; Managò, Stefano; De Angelis, Annalisa; Sirleto, Luigi; Coppola, Giuseppe; De Luca, Anna Chiara

    2015-01-01

    A full label-free morphological and biochemical characterization is desirable to select spermatozoa during preparation for artificial insemination. In order to study these fundamental parameters, we take advantage of two attractive techniques: digital holography (DH) and Raman spectroscopy (RS). DH presents new opportunities for studying morphological aspect of cells and tissues non-invasively, quantitatively and without the need for staining or tagging, while RS is a very specific technique allowing the biochemical analysis of cellular components with a spatial resolution in the sub-micrometer range. In this paper, morphological and biochemical bovine sperm cell alterations were studied using these techniques. In addition, a complementary DH and RS study was performed to identify X- and Y-chromosome-bearing sperm cells. We demonstrate that the two techniques together are a powerful and highly efficient tool elucidating some important criterions for sperm morphological selection and sex-identification, overcoming many of the limitations associated with existing protocols. PMID:25836358

  4. Label-free imaging of arterial cells and extracellular matrix using a multimodal CARS microscope

    Science.gov (United States)

    Wang, Han-Wei; Le, Thuc T.; Cheng, Ji-Xin

    2008-04-01

    A multimodal nonlinear optical imaging system that integrates coherent anti-Stokes Raman scattering (CARS), sum-frequency generation (SFG), and two-photon excitation fluorescence (TPEF) on the same platform was developed and applied to visualize single cells and extracellular matrix in fresh carotid arteries. CARS signals arising from CH 2-rich membranes allowed visualization of endothelial cells and smooth muscle cells of the arterial wall. Additionally, CARS microscopy allowed vibrational imaging of elastin and collagen fibrils which are also rich in CH 2 bonds. The extracellular matrix organization was further confirmed by TPEF signals arising from elastin's autofluorescence and SFG signals arising from collagen fibrils' non-centrosymmetric structure. Label-free imaging of significant components of arterial tissues suggests the potential application of multimodal nonlinear optical microscopy to monitor onset and progression of arterial diseases.

  5. Old tree with new shoots: silver nanoparticles for label-free and colorimetric mercury ions detection

    Science.gov (United States)

    Gao, Shuyan; Jia, Xiaoxia; Chen, Yanli

    2013-01-01

    Mercury in the environment from global mercury emissions as well as various forms of contamination poses severe threats to both human health and the environment. Long-term exposure to high levels of Hg-based toxins results in serious and irreversible damage of the central nervous system and other organs. Therefore, the development of effective sensing systems for mercury detection becomes an increasing demand. In this article, a yogurt-mediated silver nanostructure is reported to be unprecedentedly used in the naked-eye and label-free detection of mercury. The method relies on the redox reaction resulting from the electrode potential difference between Ag+/Ag (0.7996 V) and Hg2+/Hg2 2+ (0.920 V) that makes colorless Hg2+ ions which oxidize colored silver nanoparticle (AgNP) to colorless Ag+. The labor-intensive modification of AgNPs and expensive labeling are avoided, and the traditional AuNPs are substituted by AgNPs in this Hg2+ ions sensing platform, which makes it facile, low-cost, and particularly useful for home, clinic, or field applications as well as resource-limited conditions. This sensing system achieves a detection limit as low as 10 nM, lower than the toxicity level of Hg2+ ions in drinking water (30 nM) defined by World Health Organization, and exhibits excellent selectivity, largely free from the matrix effect of the real water samples. This visual label-free Hg2+ ions sensing motif shows great promise for sensing Hg2+ ions in terms of sensitivity, selectivity, cost, and maneuverability. It is also a good example for the organic combination of green chemistry and functional materials, which may trigger interest in furthering biosystems for environmental science applications.

  6. Novel label-free biosensing technology for monitoring of aqueous solutions (Conference Presentation)

    Science.gov (United States)

    Kehl, Florian; Bielecki, Robert; Follonier, Stephane; Dorokhin, Denis

    2016-03-01

    Waste water, drinking water and other industrial water sources are more and more/increasingly polluted with a large variety of contaminants, such as pesticides or residuals of pharmaceuticals. These compounds can impact human and animal organisms and lead to serious health issues. Today, in order to analyze the presence and quantity of the abovementioned micropollutants, samples are typically sent to specialized centralized laboratories and their processing may take up to several days. In order to meet the demand for continuous and consistent monitoring of aqueous solutions we propose a novel label-free technology system comprising proprietary chip and reader device designs. The core of the system is constituted by a planar-grated-waveguide (PGW) chip. Label-free biosensors, based on PGWs are sensitive to effective refractive index changes caused by the adsorption of biomolecules (micropollutants) onto the sensor surface or due to refractive index changes of the bulk solution. The presented reader device operates with a novel readout concept based on a scanning MEMS mirror for the angular interrogation of input grating couplers at a high repetition rate. The reader has fully integrated optics, electronics and fluidics and at the same time consumes limited energy (portable, field use ready). In the recent experiments, the effectiveness of the technology has been demonstrated with various liquids and bioassays showing (i) an excellent refractometric sensitivity with a limit of detection towards effective refractive index changes of ▵neff < 2 x 10-7, and (ii) the capability to perform affinity measurements for large (<150 kDa) and small (<250 Da) molecules.

  7. Label-free electrochemical immunosensor based on cerium oxide nanowires for Vibrio cholerae O1 detection

    International Nuclear Information System (INIS)

    Tam, Phuong Dinh; Thang, Cao Xuan

    2016-01-01

    This paper developed a label-free immunosensor based on cerium oxide nanowire for Vibrio cholerae O1 detection application. The CeO 2 nanowires were synthesized by hydrothermal reaction. The immobilization of Anti-V. cholerae O1 onto CeO 2 nanowire-deposited sensor was performed via an amino ester, which was created by using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, and sulfo-N-hydroxysuccinimide. The electrochemical responses of the immunosensor were studied by electrochemical impedance spectroscopy with [Fe (CN) 6 ] 3−/4− as redox probe. A linear response in electron transfer resistance for cell of V. cholerae O1 concentration was found in the range of 1.0 × 10 2 CFU/mL to 1.0 × 10 4 CFU/mL. The detection limit of the immunosensor was 1.0 × 10 2 CFU/mL. The immunosensor sensitivity was 56.82 Ω/CFU·mL −1 . Furthermore, the parameters affecting immunosensor response were also investigated, as follows: pH value, immunoreaction time, incubation temperature, and anti-V. cholerae O1 concentration. - Highlights: • A label-free immunosensor based on cerium oxide nanowire for Vibrio cholerae O1 detection application was developed. • A linear response was found in the range of 1.0 × 10 2 CFU/mL to 1.0 × 10 4 CFU/mL. • The detection limit of the immunosensor was 1.0 × 10 2 CFU/mL. • The immunosensor sensitivity was 56.82 Ω/CFU.mL −1 .

  8. Old tree with new shoots: silver nanoparticles for label-free and colorimetric mercury ions detection

    International Nuclear Information System (INIS)

    Gao Shuyan; Jia Xiaoxia; Chen Yanli

    2013-01-01

    Mercury in the environment from global mercury emissions as well as various forms of contamination poses severe threats to both human health and the environment. Long-term exposure to high levels of Hg-based toxins results in serious and irreversible damage of the central nervous system and other organs. Therefore, the development of effective sensing systems for mercury detection becomes an increasing demand. In this article, a yogurt-mediated silver nanostructure is reported to be unprecedentedly used in the naked-eye and label-free detection of mercury. The method relies on the redox reaction resulting from the electrode potential difference between Ag + /Ag (0.7996 V) and Hg 2+ /Hg 2 2+ (0.920 V) that makes colorless Hg 2+ ions which oxidize colored silver nanoparticle (AgNP) to colorless Ag+. The labor-intensive modification of AgNPs and expensive labeling are avoided, and the traditional AuNPs are substituted by AgNPs in this Hg 2+ ions sensing platform, which makes it facile, low-cost, and particularly useful for home, clinic, or field applications as well as resource-limited conditions. This sensing system achieves a detection limit as low as 10 nM, lower than the toxicity level of Hg 2+ ions in drinking water (30 nM) defined by World Health Organization, and exhibits excellent selectivity, largely free from the matrix effect of the real water samples. This visual label-free Hg 2+ ions sensing motif shows great promise for sensing Hg 2+ ions in terms of sensitivity, selectivity, cost, and maneuverability. It is also a good example for the organic combination of green chemistry and functional materials, which may trigger interest in furthering biosystems for environmental science applications.

  9. Old tree with new shoots: silver nanoparticles for label-free and colorimetric mercury ions detection

    Energy Technology Data Exchange (ETDEWEB)

    Gao Shuyan, E-mail: shuyangao@htu.cn; Jia Xiaoxia; Chen Yanli [Henan Normal University, College of Chemistry and Environmental Science (China)

    2013-01-15

    Mercury in the environment from global mercury emissions as well as various forms of contamination poses severe threats to both human health and the environment. Long-term exposure to high levels of Hg-based toxins results in serious and irreversible damage of the central nervous system and other organs. Therefore, the development of effective sensing systems for mercury detection becomes an increasing demand. In this article, a yogurt-mediated silver nanostructure is reported to be unprecedentedly used in the naked-eye and label-free detection of mercury. The method relies on the redox reaction resulting from the electrode potential difference between Ag{sup +}/Ag (0.7996 V) and Hg{sup 2+}/Hg{sub 2}{sup 2+} (0.920 V) that makes colorless Hg{sup 2+} ions which oxidize colored silver nanoparticle (AgNP) to colorless Ag+. The labor-intensive modification of AgNPs and expensive labeling are avoided, and the traditional AuNPs are substituted by AgNPs in this Hg{sup 2+} ions sensing platform, which makes it facile, low-cost, and particularly useful for home, clinic, or field applications as well as resource-limited conditions. This sensing system achieves a detection limit as low as 10 nM, lower than the toxicity level of Hg{sup 2+} ions in drinking water (30 nM) defined by World Health Organization, and exhibits excellent selectivity, largely free from the matrix effect of the real water samples. This visual label-free Hg{sup 2+} ions sensing motif shows great promise for sensing Hg{sup 2+} ions in terms of sensitivity, selectivity, cost, and maneuverability. It is also a good example for the organic combination of green chemistry and functional materials, which may trigger interest in furthering biosystems for environmental science applications.

  10. Label free sensing of creatinine using a 6 GHz CMOS near-field dielectric immunosensor.

    Science.gov (United States)

    Guha, S; Warsinke, A; Tientcheu, Ch M; Schmalz, K; Meliani, C; Wenger, Ch

    2015-05-07

    In this work we present a CMOS high frequency direct immunosensor operating at 6 GHz (C-band) for label free determination of creatinine. The sensor is fabricated in standard 0.13 μm SiGe:C BiCMOS process. The report also demonstrates the ability to immobilize creatinine molecules on a Si3N4 passivation layer of the standard BiCMOS/CMOS process, therefore, evading any further need of cumbersome post processing of the fabricated sensor chip. The sensor is based on capacitive detection of the amount of non-creatinine bound antibodies binding to an immobilized creatinine layer on the passivated sensor. The chip bound antibody amount in turn corresponds indirectly to the creatinine concentration used in the incubation phase. The determination of creatinine in the concentration range of 0.88-880 μM is successfully demonstrated in this work. A sensitivity of 35 MHz/10 fold increase in creatinine concentration (during incubation) at the centre frequency of 6 GHz is gained by the immunosensor. The results are compared with a standard optical measurement technique and the dynamic range and sensitivity is of the order of the established optical indication technique. The C-band immunosensor chip comprising an area of 0.3 mm(2) reduces the sensing area considerably, therefore, requiring a sample volume as low as 2 μl. The small analyte sample volume and label free approach also reduce the experimental costs in addition to the low fabrication costs offered by the batch fabrication technique of CMOS/BiCMOS process.

  11. Spiked proteomic standard dataset for testing label-free quantitative software and statistical methods

    Directory of Open Access Journals (Sweden)

    Claire Ramus

    2016-03-01

    Full Text Available This data article describes a controlled, spiked proteomic dataset for which the “ground truth” of variant proteins is known. It is based on the LC-MS analysis of samples composed of a fixed background of yeast lysate and different spiked amounts of the UPS1 mixture of 48 recombinant proteins. It can be used to objectively evaluate bioinformatic pipelines for label-free quantitative analysis, and their ability to detect variant proteins with good sensitivity and low false discovery rate in large-scale proteomic studies. More specifically, it can be useful for tuning software tools parameters, but also testing new algorithms for label-free quantitative analysis, or for evaluation of downstream statistical methods. The raw MS files can be downloaded from ProteomeXchange with identifier http://www.ebi.ac.uk/pride/archive/projects/PXD001819. Starting from some raw files of this dataset, we also provide here some processed data obtained through various bioinformatics tools (including MaxQuant, Skyline, MFPaQ, IRMa-hEIDI and Scaffold in different workflows, to exemplify the use of such data in the context of software benchmarking, as discussed in details in the accompanying manuscript [1]. The experimental design used here for data processing takes advantage of the different spike levels introduced in the samples composing the dataset, and processed data are merged in a single file to facilitate the evaluation and illustration of software tools results for the detection of variant proteins with different absolute expression levels and fold change values.

  12. A comprehensive evaluation of popular proteomics software workflows for label-free proteome quantification and imputation.

    Science.gov (United States)

    Välikangas, Tommi; Suomi, Tomi; Elo, Laura L

    2017-05-31

    Label-free mass spectrometry (MS) has developed into an important tool applied in various fields of biological and life sciences. Several software exist to process the raw MS data into quantified protein abundances, including open source and commercial solutions. Each software includes a set of unique algorithms for different tasks of the MS data processing workflow. While many of these algorithms have been compared separately, a thorough and systematic evaluation of their overall performance is missing. Moreover, systematic information is lacking about the amount of missing values produced by the different proteomics software and the capabilities of different data imputation methods to account for them.In this study, we evaluated the performance of five popular quantitative label-free proteomics software workflows using four different spike-in data sets. Our extensive testing included the number of proteins quantified and the number of missing values produced by each workflow, the accuracy of detecting differential expression and logarithmic fold change and the effect of different imputation and filtering methods on the differential expression results. We found that the Progenesis software performed consistently well in the differential expression analysis and produced few missing values. The missing values produced by the other software decreased their performance, but this difference could be mitigated using proper data filtering or imputation methods. Among the imputation methods, we found that the local least squares (lls) regression imputation consistently increased the performance of the software in the differential expression analysis, and a combination of both data filtering and local least squares imputation increased performance the most in the tested data sets. © The Author 2017. Published by Oxford University Press.

  13. Label-free electrochemical immunosensor based on cerium oxide nanowires for Vibrio cholerae O1 detection

    Energy Technology Data Exchange (ETDEWEB)

    Tam, Phuong Dinh, E-mail: phuongdinhtam@gmail.com; Thang, Cao Xuan, E-mail: thang.caoxuan@hust.edu.vn

    2016-01-01

    This paper developed a label-free immunosensor based on cerium oxide nanowire for Vibrio cholerae O1 detection application. The CeO{sub 2} nanowires were synthesized by hydrothermal reaction. The immobilization of Anti-V. cholerae O1 onto CeO{sub 2} nanowire-deposited sensor was performed via an amino ester, which was created by using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, and sulfo-N-hydroxysuccinimide. The electrochemical responses of the immunosensor were studied by electrochemical impedance spectroscopy with [Fe (CN) {sub 6}] {sup 3−/4−} as redox probe. A linear response in electron transfer resistance for cell of V. cholerae O1 concentration was found in the range of 1.0 × 10{sup 2} CFU/mL to 1.0 × 10{sup 4} CFU/mL. The detection limit of the immunosensor was 1.0 × 10{sup 2} CFU/mL. The immunosensor sensitivity was 56.82 Ω/CFU·mL{sup −1}. Furthermore, the parameters affecting immunosensor response were also investigated, as follows: pH value, immunoreaction time, incubation temperature, and anti-V. cholerae O1 concentration. - Highlights: • A label-free immunosensor based on cerium oxide nanowire for Vibrio cholerae O1 detection application was developed. • A linear response was found in the range of 1.0 × 10{sup 2} CFU/mL to 1.0 × 10{sup 4} CFU/mL. • The detection limit of the immunosensor was 1.0 × 10{sup 2} CFU/mL. • The immunosensor sensitivity was 56.82 Ω/CFU.mL{sup −1}.

  14. Large area, label-free imaging of extracellular matrix using telecentricity

    Science.gov (United States)

    Visbal Onufrak, Michelle A.; Konger, Raymond L.; Kim, Young L.

    2017-02-01

    Subtle alterations in stromal tissue structures and organizations within the extracellular matrix (ECM) have been observed in several types of tissue abnormalities, including early skin cancer and wounds. Current microscopic imaging methods often lack the ability to accurately determine the extent of malignancy over a large area, due to their limited field of view. In this research we focus on the development of simple mesoscopic (i.e. between microscopic and macroscopic) biomedical imaging device for non-invasive assessment of ECM alterations over a large, heterogeneous area. In our technology development, a telecentric lens, commonly used in machine vision systems but rarely used in biomedical imaging, serves as a key platform to visualize alterations in tissue microenvironments in a label-free manner over a clinically relevant area. In general, telecentric imaging represents a simple, alternative method for reducing unwanted scattering or diffuse light caused by the highly anisotropic scattering properties of biological tissue. In particular, under telecentric imaging the light intensity backscattered from biological tissue is mainly sensitive to the scattering anisotropy factor, possibly associated with the ECM. We demonstrate the inherent advantages of combining telecentric lens systems with hyperspectral imaging for providing optical information of tissue scattering in biological tissue of murine models, as well as light absorption of hemoglobin in blood vessel tissue phantoms. Thus, we envision that telecentric imaging could potentially serve for simple site-specific, tissue-based assessment of stromal alterations over a clinically relevant field of view in a label-free manner, for studying diseases associated with disruption of homeostasis in ECM.

  15. Label-free SnO2 nanowire FET biosensor for protein detection

    Science.gov (United States)

    Jakob, Markus H.; Dong, Bo; Gutsch, Sebastian; Chatelle, Claire; Krishnaraja, Abinaya; Weber, Wilfried; Zacharias, Margit

    2017-06-01

    Novel tin oxide field-effect-transistors (SnO2 NW-FET) for pH and protein detection applicable in the healthcare sector are reported. With a SnO2 NW-FET the proof-of-concept of a bio-sensing device is demonstrated using the carrier transport control of the FET channel by a (bio-) liquid modulated gate. Ultra-thin Al2O3 fabricated by a low temperature atomic layer deposition (ALD) process represents a sensitive layer to H+ ions safeguarding the nanowire at the same time. Successful pH sensitivity is demonstrated for pH ranging from 3 to 10. For protein detection, the SnO2 NW-FET is functionalized with a receptor molecule which specifically interacts with the protein of interest to be detected. The feasibility of this approach is demonstrated via the detection of a biotinylated protein using a NW-FET functionalized with streptavidin. An immediate label-free electronic read-out of the signal is shown. The well-established Enzyme-Linked Immunosorbent Assay (ELISA) method is used to determine the optimal experimental procedure which would enable molecular binding events to occur while being compatible with a final label-free electronic read-out on a NW-FET. Integration of the bottom-up fabricated SnO2 NW-FET pH- and biosensor into a microfluidic system (lab-on-a-chip) allows the automated analysis of small volumes in the 400 μl range as would be desired in portable on-site point-of-care (POC) devices for medical diagnosis.

  16. Label-free electrochemiluminescence biosensor for ultrasensitive detection of telomerase activity in HeLa cells based on extension reaction and intercalation of Ru(phen)3 (2.).

    Science.gov (United States)

    Lin, Yue; Yang, Linlin; Yue, Guiyin; Chen, Lifen; Qiu, Bin; Guo, Longhua; Lin, Zhenyu; Chen, Guonan

    2016-10-01

    Telomerase is one of the most common markers of human malignant tumors, such as uterine, stomach, esophageal, breast, colorectal, laryngeal squamous cell, thyroid, bladder, and so on. It is necessary to develop some sensitive but convenient detection methods for telomerase activity determination. In this study, a label-free and ultrasensitive electrochemiluminescence (ECL) biosensor has been fabricated to detect the activity of telomerase extracted from HeLa cells. Thiolated telomerase substrate (TS) primer was immobilized on the gold electrode surface through gold-sulfur (Au-S) interaction and then elongated by telomerase specifically. Then, it was hybridized with complementary DNA to form double-stranded DNA (dsDNA) fragments on the electrode surface, and Ru(phen)3 (2+) has been intercalated into the dsDNA grooves to act as the ECL probe. The enhanced ECL intensity has a linear relationship with the number of HeLa cells in the range of 5∼5000 and with a detection limit of 2 HeLa cells. The proposed ECL biosensor has high specificity to telomerase in the presence of common interferents. The relative standard deviations (RSDs) were HeLa cells. The proposed method provides a convenient approach for telomerase-related cancer screening or diagnosis.

  17. Pyrrolidinyl PNA polypyrrole/silver nanofoam electrode as a novel label-free electrochemical miRNA-21 biosensor.

    Science.gov (United States)

    Kangkamano, Tawatchai; Numnuam, Apon; Limbut, Warakorn; Kanatharana, Proespichaya; Vilaivan, Tirayut; Thavarungkul, Panote

    2018-04-15

    A label-free electrochemical miRNA biosensor was developed based on a pyrrolidinyl peptide nucleic acid (acpcPNA)/polypyrrole (PPy)/silver nanofoam (AgNF) modified electrode. The AgNF was electrodeposited as redox indicator on a gold electrode, which was then functionalized with an electropolymerized layer of PPy, a conducting polymer, to immobilize the PNA probes. The fabrication process was investigated by electrochemical impedance spectroscopy. The biosensor was used to detect miRNA-21, a biomarker abnormally expressed in most cancers. The signal was monitored by the change in current of the AgNF redox reaction before and after hybridization using cyclic voltammetry. Two PNA probe lengths were investigated and the longer probe exhibited a better performance. Nucleotide overhangs on the electrode side affected the signal more than overhangs on the solution side due to the greater insulation of the sensing surface. Under optimal conditions, the electrochemical signal was proportional to miRNA-21 concentrations between 0.20fM and 1.0nM, with a very low detection limit of 0.20fM. The biosensor showed a high specificity which could discriminate between complementary, single-, doubled-base mismatched, and non-complementary targets. Three out of the seven tested plasma samples provided detectable concentrations (63 ± 4, 111 ± 4 and 164 ± 7fM). The sensor also showed good recoveries (81-119%). The results indicated the possibilities of this biosensor for analysis without RNA extraction and/or amplification, making the sensor potentially useful for both the prognosis and diagnosis of cancer in clinical application. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. Detection of Target ssDNA Using a Microfabricated Hall Magnetometer with Correlated Optical Readout

    Directory of Open Access Journals (Sweden)

    Steven M. Hira

    2012-01-01

    Full Text Available Sensing biological agents at the genomic level, while enhancing the response time for biodetection over commonly used, optics-based techniques such as nucleic acid microarrays or enzyme-linked immunosorbent assays (ELISAs, is an important criterion for new biosensors. Here, we describe the successful detection of a 35-base, single-strand nucleic acid target by Hall-based magnetic transduction as a mimic for pathogenic DNA target detection. The detection platform has low background, large signal amplification following target binding and can discriminate a single, 350 nm superparamagnetic bead labeled with DNA. Detection of the target sequence was demonstrated at 364 pM (<2 target DNA strands per bead target DNA in the presence of 36 μM nontarget (noncomplementary DNA (<10 ppm target DNA using optical microscopy detection on a GaAs Hall mimic. The use of Hall magnetometers as magnetic transduction biosensors holds promise for multiplexing applications that can greatly improve point-of-care (POC diagnostics and subsequent medical care.

  19. Suppression of resonance Raman scattering via ground state depletion towards sub-diffraction-limited label-free microscopy

    NARCIS (Netherlands)

    Rieger, S.; Fischedick, M.; Boller, Klaus J.; Fallnich, Carsten

    2016-01-01

    We report on the first experimental demonstration of the suppression of spontaneous Raman scattering via ground state depletion. The concept of Raman suppression can be used to achieve sub-diffraction-limited resolution in label-free microscopy by exploiting spatially selective signal suppression

  20. Electrokinetic label-free screening chip: a marriage of multiplexing and high throughput analysis using surface plasmon resonance imaging

    NARCIS (Netherlands)

    Krishnamoorthy, G.; Carlen, Edwin; Bomer, Johan G.; Wijnperle, Daniël; de Boer, Hans L.; van den Berg, Albert; Schasfoort, Richardus B.M.

    2010-01-01

    We present an electrokinetic label-free biomolecular screening chip (Glass/PDMS) to screen up to 10 samples simultaneously using surface plasmon resonance imaging (iSPR). This approach reduces the duration of an experiment when compared to conventional experimental methods. This new device offers a

  1. Universal chitosan-assisted synthesis of Ag-including heterostructured nanocrystals for label-free in situ SERS monitoring.

    Science.gov (United States)

    Cai, Kai; Xiao, Xiaoyan; Zhang, Huan; Lu, Zhicheng; Liu, Jiawei; Li, Qin; Liu, Chen; Foda, Mohamed F; Han, Heyou

    2015-12-07

    A universal chitosan-assisted method was developed to synthesize various Ag-including heterostructured nanocrystals, in which chelation probably plays a vital role. The as-prepared Ag/Pd heterostructured nanocrystals show outstanding properties when used as bifunctional nanocomposites in label-free in situ SERS monitoring of Pd-catalyzed reaction.

  2. Applying label-free dynamic mass redistribution assay for studying endogenous FPR1 receptor signalling in human neutrophils

    DEFF Research Database (Denmark)

    Christensen, Hanna B; Gloriam, David E; Pedersen, Daniel Sejer

    2017-01-01

    INTRODUCTION: The label-free dynamic mass redistribution-based assay (DMR) is a powerful method for studying signalling pathways of G protein-coupled receptors (GPCRs). Herein we present the label-free DMR assay as a robust readout for pharmacological characterization of formyl peptide receptors...... (FPRs) in human neutrophils. METHODS: Neutrophils were isolated from fresh human blood and their responses to FPR1 and FPR2 agonists, i.e. compound 43, fMLF and WKYMVm were measured in a label-free DMR assay using Epic Benchtop System from Corning®. Obtained DMR traces were used to calculate agonist...... potencies. RESULTS: The potencies (pEC50) of fMLF, WKYMVm and compound 43, determined on human neutrophils using the label-free DMR assay were 8.63, 7.76 and 5.92, respectively. The DMR response to fMLF, but not WKYMVm and compound 43 could be blocked by the FPR1-specific antagonist cyclosporin H...

  3. Label-Free Detection of Glycan-Protein Interactions for Array Development by Surface-Enhanced Raman Spectroscopy (SERS)

    NARCIS (Netherlands)

    Li, Xiuru; Martin, Sharon J H; Chinoy, Zoeisha S; Liu, Lin; Rittgers, Brandon; Dluhy, Richard A; Boons, Geert-Jan

    2016-01-01

    A glyco-array platform has been developed, in which glycans are attached to plasmonic nanoparticles through strain-promoted azide-alkyne cycloaddition. Glycan-protein binding events can then be detected in a label-free manner employing surface-enhanced Raman spectroscopy (SERS). As proof of concept,

  4. Functionalizable low-fouling coatings for label-free biosensing in complex biological media: advances and applications

    Czech Academy of Sciences Publication Activity Database

    Vaisocherová, Hana; Brynda, Eduard; Homola, Jiří

    2015-01-01

    Roč. 407, č. 14 (2015), s. 3927-3953 ISSN 1618-2642 R&D Projects: GA ČR(CZ) GBP205/12/G118 Institutional support: RVO:67985882 ; RVO:61389013 Keywords : Label-free optical biosensors * Polymer brushes * Blood plasma Subject RIV: JA - Electronics ; Optoelectronics, Electrical Engineering Impact factor: 3.125, year: 2015

  5. The goat (Capra hircus) mammary gland secretory tissue proteome as influenced by weight loss: A study using label free proteomics

    DEFF Research Database (Denmark)

    Hernandez Castellano, Lorenzo E; Ferreira, Ana M.; Nanni, Paolo

    2016-01-01

    Seasonal weight loss (SWL) is a significant limitation to animal production. Breeds that have evolved in harsh climates have acquired tolerance to SWL through selection. Herein, label free proteomics was used to characterize the effects of SWL in two goat breeds with different levels of adaptatio...

  6. A Label-Free and Sensitive Fluorescent Qualitative Assay for Bisphenol A Based on Rolling Circle Amplification/Exonuclease III-Combined Cascade Amplification.

    Science.gov (United States)

    Li, Xia; Song, Juan; Xue, Qing-Wang; You, Fu-Heng; Lu, Xia; Kong, Yan-Cong; Ma, Shu-Yi; Jiang, Wei; Li, Chen-Zhong

    2016-10-21

    Bisphenol A (BPA) detection in drinking water and food packaging materials has attracted much attention since the discovery that BPA can interfere with normal physiological processes and cause adverse health effects. Here, we constructed a label-free aptamer fluorescent assay for selective and sensitive detection of BPA based on the rolling circle amplification (RCA)/Exonuclease III (Exo III)-combined cascade amplification strategy. First, the duplex DNA probe (RP) with anti-BPA aptamer and trigger sequence was designed for BPA recognition and signal amplification. Next, under the action of BPA, the trigger probe was liberated from RP to initiate RCA reaction as primary amplification. Subsequently, the RCA products were used to trigger Exo III assisted secondary amplification with the help of hairpin probes, producing plenty of "G-quadruplex" in lantern-like structures. Finally, the continuously enriched "G-quadruplex lanterns" were lightened by zinc(II)-protoporphyrin IX (ZnPPIX) generating enhanced fluorescence signals. By integrating the primary RCA and secondary Exo III mediated cascade amplification strategy, this method displayed an excellent sensitivity with the detection limits of 5.4 × 10 -17 M. In addition, the anti-BPA aptamer exhibits high recognition ability with BPA, guaranteeing the specificity of detection. The reporter signal probe (G-quadruplex with ZnPPIX) provides a label-free fluorescence signals readout without complicated labeling procedures, making the method simple in design and cost-effective in operation. Moreover, environmental samples analysis was also performed, suggesting that our strategy was reliable and had a great potential application in environmental monitoring.

  7. A Label-Free and Sensitive Fluorescent Qualitative Assay for Bisphenol A Based on Rolling Circle Amplification/Exonuclease III-Combined Cascade Amplification

    Directory of Open Access Journals (Sweden)

    Xia Li

    2016-10-01

    Full Text Available Bisphenol A (BPA detection in drinking water and food packaging materials has attracted much attention since the discovery that BPA can interfere with normal physiological processes and cause adverse health effects. Here, we constructed a label-free aptamer fluorescent assay for selective and sensitive detection of BPA based on the rolling circle amplification (RCA/Exonuclease III (Exo III-combined cascade amplification strategy. First, the duplex DNA probe (RP with anti-BPA aptamer and trigger sequence was designed for BPA recognition and signal amplification. Next, under the action of BPA, the trigger probe was liberated from RP to initiate RCA reaction as primary amplification. Subsequently, the RCA products were used to trigger Exo III assisted secondary amplification with the help of hairpin probes, producing plenty of “G-quadruplex” in lantern-like structures. Finally, the continuously enriched “G-quadruplex lanterns” were lightened by zinc(II-protoporphyrin IX (ZnPPIX generating enhanced fluorescence signals. By integrating the primary RCA and secondary Exo III mediated cascade amplification strategy, this method displayed an excellent sensitivity with the detection limits of 5.4 × 10−17 M. In addition, the anti-BPA aptamer exhibits high recognition ability with BPA, guaranteeing the specificity of detection. The reporter signal probe (G-quadruplex with ZnPPIX provides a label-free fluorescence signals readout without complicated labeling procedures, making the method simple in design and cost-effective in operation. Moreover, environmental samples analysis was also performed, suggesting that our strategy was reliable and had a great potential application in environmental monitoring.

  8. RNF111/Arkadia is a SUMO-targeted ubiquitin ligase that facilitates the DNA damage response

    DEFF Research Database (Denmark)

    Poulsen, Sara L; Hansen, Rebecca K; Wagner, Sebastian A

    2013-01-01

    nonproteolytic, K63-linked ubiquitylation of SUMOylated target proteins. We demonstrate that RNF111 promoted ubiquitylation of SUMOylated XPC (xeroderma pigmentosum C) protein, a central DNA damage recognition factor in nucleotide excision repair (NER) extensively regulated by ultraviolet (UV...

  9. High-affinity triplex targeting of double stranded DNA using chemically modified peptide nucleic acid oligomers

    DEFF Research Database (Denmark)

    Hansen, Mads E; Bentin, Thomas; Nielsen, Peter E

    2009-01-01

    While sequence-selective dsDNA targeting by triplex forming oligonucleotides has been studied extensively, only very little is known about the properties of PNA-dsDNA triplexes-mainly due to the competing invasion process. Here we show that when appropriately modified using pseudoisocytosine subs...

  10. Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease.

    Science.gov (United States)

    Anders, Carolin; Niewoehner, Ole; Duerst, Alessia; Jinek, Martin

    2014-09-25

    The CRISPR-associated protein Cas9 is an RNA-guided endonuclease that cleaves double-stranded DNA bearing sequences complementary to a 20-nucleotide segment in the guide RNA. Cas9 has emerged as a versatile molecular tool for genome editing and gene expression control. RNA-guided DNA recognition and cleavage strictly require the presence of a protospacer adjacent motif (PAM) in the target DNA. Here we report a crystal structure of Streptococcus pyogenes Cas9 in complex with a single-molecule guide RNA and a target DNA containing a canonical 5'-NGG-3' PAM. The structure reveals that the PAM motif resides in a base-paired DNA duplex. The non-complementary strand GG dinucleotide is read out via major-groove interactions with conserved arginine residues from the carboxy-terminal domain of Cas9. Interactions with the minor groove of the PAM duplex and the phosphodiester group at the +1 position in the target DNA strand contribute to local strand separation immediately upstream of the PAM. These observations suggest a mechanism for PAM-dependent target DNA melting and RNA-DNA hybrid formation. Furthermore, this study establishes a framework for the rational engineering of Cas9 enzymes with novel PAM specificities.

  11. Label-free proteome profiling reveals developmental-dependent patterns in young barley grains.

    Science.gov (United States)

    Kaspar-Schoenefeld, Stephanie; Merx, Kathleen; Jozefowicz, Anna Maria; Hartmann, Anja; Seiffert, Udo; Weschke, Winfriede; Matros, Andrea; Mock, Hans-Peter

    2016-06-30

    Due to its importance as a cereal crop worldwide, high interest in the determination of factors influencing barley grain quality exists. This study focusses on the elucidation of protein networks affecting early grain developmental processes. NanoLC-based separation coupled to label-free MS detection was applied to gain insights into biochemical processes during five different grain developmental phases (pre-storage until storage phase, 3days to 16days after flowering). Multivariate statistics revealed two distinct developmental patterns during the analysed grain developmental phases: proteins showed either highest abundance in the middle phase of development - in the transition phase - or at later developmental stages - within the storage phase. Verification of developmental patterns observed by proteomic analysis was done by applying hypothesis-driven approaches, namely Western Blot analysis and enzyme assays. High general metabolic activity of the grain with regard to protein synthesis, cell cycle regulation, defence against oxidative stress, and energy production via photosynthesis was observed in the transition phase. Proteins upregulated in the storage phase are related towards storage protein accumulation, and interestingly to the defence of storage reserves against pathogens. A mixed regulatory pattern for most enzymes detected in our study points to regulatory mechanisms at the level of protein isoforms. In-depth understanding of early grain developmental processes of cereal caryopses is of high importance as they influence final grain weight and quality. Our knowledge about these processes is still limited, especially on proteome level. To identify key mechanisms in early barley grain development, a label-free data-independent proteomics acquisition approach has been applied. Our data clearly show, that proteins either exhibit highest expression during cellularization and the switch to the storage phase (transition phase, 5-7 DAF), or during storage

  12. Mechanism of duplex DNA destabilization by RNA-guided Cas9 nuclease during target interrogation.

    Science.gov (United States)

    Mekler, Vladimir; Minakhin, Leonid; Severinov, Konstantin

    2017-05-23

    The prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)-associated 9 (Cas9) endonuclease cleaves double-stranded DNA sequences specified by guide RNA molecules and flanked by a protospacer adjacent motif (PAM) and is widely used for genome editing in various organisms. The RNA-programmed Cas9 locates the target site by scanning genomic DNA. We sought to elucidate the mechanism of initial DNA interrogation steps that precede the pairing of target DNA with guide RNA. Using fluorometric and biochemical assays, we studied Cas9/guide RNA complexes with model DNA substrates that mimicked early intermediates on the pathway to the final Cas9/guide RNA-DNA complex. The results show that Cas9/guide RNA binding to PAM favors separation of a few PAM-proximal protospacer base pairs allowing initial target interrogation by guide RNA. The duplex destabilization is mediated, in part, by Cas9/guide RNA affinity for unpaired segments of nontarget strand DNA close to PAM. Furthermore, our data indicate that the entry of double-stranded DNA beyond a short threshold distance from PAM into the Cas9/single-guide RNA (sgRNA) interior is hindered. We suggest that the interactions unfavorable for duplex DNA binding promote DNA bending in the PAM-proximal region during early steps of Cas9/guide RNA-DNA complex formation, thus additionally destabilizing the protospacer duplex. The mechanism that emerges from our analysis explains how the Cas9/sgRNA complex is able to locate the correct target sequence efficiently while interrogating numerous nontarget sequences associated with correct PAMs.

  13. Comparative study of label and label-free techniques using shotgun proteomics for relative protein quantification.

    Science.gov (United States)

    Sjödin, Marcus O D; Wetterhall, Magnus; Kultima, Kim; Artemenko, Konstantin

    2013-06-01

    The analytical performance of three different strategies, iTRAQ (isobaric tag for relative and absolute quantification), dimethyl labeling (DML) and label free (LF) for relative protein quantification using shotgun proteomics have been evaluated. The methods have been explored using samples containing (i) Bovine proteins in known ratios and (ii) Bovine proteins in known ratios spiked into Escherichia coli. The latter case mimics the actual conditions in a typical biological sample with a few differentially expressed proteins and a bulk of proteins with unchanged ratios. Additionally, the evaluation was performed on both QStar and LTQ-FTICR mass spectrometers. LF LTQ-FTICR was found to have the highest proteome coverage while the highest accuracy based on the artificially regulated proteins was found for DML LTQ-FTICR (54%). A varying linearity (k: 0.55-1.16, r(2): 0.61-0.96) was shown for all methods within selected dynamic ranges. All methods were found to consistently underestimate Bovine protein ratios when matrix proteins were added. However, LF LTQ-FTICR was more tolerant toward a compression effect. A single peptide was demonstrated to be sufficient for a reliable quantification using iTRAQ. A ranking system utilizing several parameters important for quantitative proteomics demonstrated that the overall performance of the five different methods was; DML LTQ-FTICR>iTRAQ QStar>LF LTQ-FTICR>DML QStar>LF QStar. Copyright © 2013 Elsevier B.V. All rights reserved.

  14. Biconically Tapered Fiber Optic Probes for Rapid Label-Free Immunoassays

    Directory of Open Access Journals (Sweden)

    John Miller

    2015-04-01

    Full Text Available We report use of U-shaped biconically tapered optical fibers (BTOF as probes for label-free immunoassays. The tapered regions of the sensors were functionalized by immobilization of immunoglobulin-G (Ig-G and tested for detection of anti-IgG at concentrations of 50 ng/mL to 50 µg/mL. Antibody-antigen reaction creates a biological nanolayer modifying the waveguide structure leading to a change in the sensor signal, which allows real-time monitoring. The kinetics of the antibody (mouse Ig-G-antigen (rabbit anti-mouse IgG reactions was studied. Hydrofluoric acid treatment makes the sensitive region thinner to enhance sensitivity, which we confirmed by experiments and simulations. The limit of detection for the sensor was estimated to be less than 50 ng/mL. Utilization of the rate of the sensor peak shift within the first few minutes of the antibody-antigen reaction is proposed as a rapid protein detection method.

  15. Label-Free QCM Immunosensor for the Detection of Ochratoxin A

    Directory of Open Access Journals (Sweden)

    Şerife Şeyda Pirinçci

    2018-04-01

    Full Text Available Ochratoxin A (OTA is a potent mycotoxin that poses a risk in food and feed moieties and subject to worldwide regulation. Laboratory-based analytical methods are traditionally employed for reliable OTA quantification, but these methods cannot provide rapid and on-site analysis, where biosensors fill this gap. In this study a label-free quartz crystal microbalance (QCM-based immunosensor for the detection of OTA, which is one of the most important small molecule contaminants, was developed by direct immobilization of OTA to amine-bearing sensor surfaces using 1-ethyl-3-(3-dimethylaminopropyl carbodiimide (EDC/N-Hydroxysuccinimide (NHS chemistry. The protein-free sensor surface enabled regeneration of sensor surface with 50 mM NaOH and 1% SDS up to 13 times without loss of performance, which would disrupt a protein-containing sensor surface. We developed a QCM immunosensor using the developed sensor surface with a 17.2–200 ng/mL detection range which can be used for on-site detection of feedstuffs.

  16. Divergent Label-free Cell Phenotypic Pharmacology of Ligands at the Overexpressed β2-Adrenergic Receptors

    Science.gov (United States)

    Ferrie, Ann M.; Sun, Haiyan; Zaytseva, Natalya; Fang, Ye

    2014-01-01

    We present subclone sensitive cell phenotypic pharmacology of ligands at the β2-adrenergic receptor (β2-AR) stably expressed in HEK-293 cells. The parental cell line was transfected with green fluorescent protein (GFP)-tagged β2-AR. Four stable subclones were established and used to profile a library of sixty-nine AR ligands. Dynamic mass redistribution (DMR) profiling resulted in a pharmacological activity map suggesting that HEK293 endogenously expresses functional Gi-coupled α2-AR and Gs-coupled β2-AR, and the label-free cell phenotypic activity of AR ligands are subclone dependent. Pathway deconvolution revealed that the DMR of epinephrine is originated mostly from the remodeling of actin microfilaments and adhesion complexes, to less extent from the microtubule networks and receptor trafficking, and certain agonists displayed different efficacy towards the cAMP-Epac pathway. We demonstrate that receptor signaling and ligand pharmacology is sensitive to the receptor expression level, and the organization of the receptor and its signaling circuitry.

  17. Label free selective detection of estriol using graphene oxide-based fluorescence sensor

    Science.gov (United States)

    Kushwaha, H. S.; Sao, Reshma; Vaish, Rahul

    2014-07-01

    Water-soluble and fluorescent Graphene oxide (GO) is biocompatible, easy, and economical to synthesize. Interestingly, GO is also capable of quenching fluorescence. On the basis of its fluorescence and quenching abilities, GO has been reported to serve as an energy acceptor in a fluorescence resonance energy transfer (FRET) sensor. GO-based FRET biosensors have been widely reported for sensing of proteins, nucleic acid, ATP (Adenosine triphosphate), etc. GO complexes with fluorescent dyes and enzymes have been used to sense metal ions. Graphene derivatives have been used for sensing endocrine-disrupting chemicals like bisphenols and chlorophenols with high sensitivity and good reproducibility. On this basis, a novel GO based fluorescent sensor has been successfully designed to detect estriol with remarkable selectivity and sensitivity. Estriol is one of the three estrogens in women and is considered to be medically important. Estriol content of maternal urine or plasma acts as an important screening marker for estimating foetal growth and development. In addition, estriol is also used as diagnostic marker for diseases like breast cancer, osteoporosis, neurodegenerative and cardiovascular diseases, insulin resistance, lupus erythematosus, endometriosis, etc. In this present study, we report for the first time a rapid, sensitive with detection limit of 1.3 nM, selective and highly biocompatible method for label free detection of estriol under physiological conditions using fluorescence assay.

  18. Label-free immunosensor based on Pd nanoplates for amperometric immunoassay of alpha-fetoprotein.

    Science.gov (United States)

    Wang, Huan; Li, He; Zhang, Yihe; Wei, Qin; Ma, Hongmin; Wu, Dan; Li, Yan; Zhang, Yong; Du, Bin

    2014-03-15

    In this paper, Pd nanoplates were used as a kind of electrode materials for fabrication of an electrochemical immunosensor, which was applied for detection of cancer biomarker alpha-fetoprotein (AFP). Thanks to the unique structure and properties of Pd nanoplates, the antibody of AFP (Ab) was effectively immobilized onto the surface of the Pd nanoplates modified glassy carbon electrode (GCE). Moreover, the good electrochemical properties of Pd nanoplates greatly improved the electronic transmission rate and enhanced the electrochemical signal, which led to an increase of the detection sensitivity. Based on the specific antibody-antigen interaction, a label-free immunosensor based on Pd nanoplates was developed for sensing of AFP. The current method allows us to detect AFP over a wide concentration range from 0.01 to 75.0 ng/mL with a detection limit of 4 pg/mL. The proposed immunosensor has been used to determine AFP in human serum with satisfactory results. © 2013 Elsevier B.V. All rights reserved.

  19. Minimizing technical variation during sample preparation prior to label-free quantitative mass spectrometry.

    Science.gov (United States)

    Scheerlinck, E; Dhaenens, M; Van Soom, A; Peelman, L; De Sutter, P; Van Steendam, K; Deforce, D

    2015-12-01

    Sample preparation is the crucial starting point to obtain high-quality mass spectrometry data and can be divided into two main steps in a bottom-up proteomics approach: cell/tissue lysis with or without detergents and a(n) (in-solution) digest comprising denaturation, reduction, alkylation, and digesting of the proteins. Here, some important considerations, among others, are that the reagents used for sample preparation can inhibit the digestion enzyme (e.g., 0.1% sodium dodecyl sulfate [SDS] and 0.5 M guanidine HCl), give rise to ion suppression (e.g., polyethylene glycol [PEG]), be incompatible with liquid chromatography-tandem mass spectrometry (LC-MS/MS) (e.g., SDS), and can induce additional modifications (e.g., urea). Taken together, all of these irreproducible effects are gradually becoming a problem when label-free quantitation of the samples is envisioned such as during the increasingly popular high-definition mass spectrometry (HDMS(E)) and sequential window acquisition of all theoretical fragment ion spectra (SWATH) data-independent acquisition strategies. Here, we describe the detailed validation of a reproducible method with sufficient protein yield for sample preparation without any known LC-MS/MS interfering substances by using 1% sodium deoxycholate (SDC) during both cell lysis and in-solution digest. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  20. Detection of Myoglobin with an Open-Cavity-Based Label-Free Photonic Crystal Biosensor.

    Science.gov (United States)

    Zhang, Bailin; Tamez-Vela, Juan Manuel; Solis, Steven; Bustamante, Gilbert; Peterson, Ralph; Rahman, Shafiqur; Morales, Andres; Tang, Liang; Ye, Jing Yong

    2013-01-01

    The label-free detection of one of the cardiac biomarkers, myoglobin, using a photonic-crystal-based biosensor in a total-internal-reflection configuration (PC-TIR) is presented in this paper. The PC-TIR sensor possesses a unique open optical microcavity that allows for several key advantages in biomolecular assays. In contrast to a conventional closed microcavity, the open configuration allows easy functionalization of the sensing surface for rapid biomolecular binding assays. Moreover, the properties of PC structures make it easy to be designed and engineered for operating at any optical wavelength. Through fine design of the photonic crystal structure, biochemical modification of the sensor surface, and integration with a microfluidic system, we have demonstrated that the detection sensitivity of the sensor for myoglobin has reached the clinically significant concentration range, enabling potential usage of this biosensor for diagnosis of acute myocardial infarction. The real-time response of the sensor to the myoglobin binding may potentially provide point-of-care monitoring of patients and treatment effects.

  1. Cell Surface Proteome of Dental Pulp Stem Cells Identified by Label-Free Mass Spectrometry.

    Directory of Open Access Journals (Sweden)

    Christian Niehage

    Full Text Available Multipotent mesenchymal stromal cells (MSCs are promising tools for regenerative medicine. They can be isolated from different sources based on their plastic-adherence property. The identification of reliable cell surface markers thus becomes the Holy Grail for their prospective isolation. Here, we determine the cell surface proteomes of human dental pulp-derived MSCs isolated from single donors after culture expansion in low (2% or high (10% serum-containing media. Cell surface proteins were tagged on intact cells using cell impermeable, cleavable sulfo-NHS-SS-biotin, which allows their enrichment by streptavidin pull-down. For the proteomic analyses, we first compared label-free methods to analyze cell surface proteomes i.e. composition, enrichment and proteomic differences, and we developed a new mathematical model to determine cell surface protein enrichment using a combinatorial gene ontology query. Using this workflow, we identified 101 cluster of differentiation (CD markers and 286 non-CD cell surface proteins. Based on this proteome profiling, we identified 14 cell surface proteins, which varied consistently in abundance when cells were cultured under low or high serum conditions. Collectively, our analytical methods provide a basis for identifying the cell surface proteome of dental pulp stem cells isolated from single donors and its evolution during culture or differentiation. Our data provide a comprehensive cell surface proteome for the precise identification of dental pulp-derived MSC populations and their isolation for potential therapeutic intervention.

  2. Label-Free Raman Hyperspectral Imaging of Single Cells Cultured on Polymer Substrates.

    Science.gov (United States)

    Sinjab, Faris; Sicilia, Giovanna; Shipp, Dustin W; Marlow, Maria; Notingher, Ioan

    2017-12-01

    While Raman hyperspectral imaging has been widely used for label-free mapping of biomolecules in cells, these measurements require the cells to be cultured on weakly Raman scattering substrates. However, many applications in biological sciences and engineering require the cells to be cultured on polymer substrates that often generate large Raman scattering signals. Here, we discuss the theoretical limits of the signal-to-noise ratio in the Raman spectra of cells in the presence of polymer signals and how optical aberrations may affect these measurements. We show that Raman spectra of cells cultured on polymer substrates can be obtained using automatic subtraction of the polymer signals and demonstrate the capabilities of these methods in two important applications: tissue engineering and in vitro toxicology screening of drugs. Apart from their scientific and technological importance, these applications are examples of the two most common measurement configurations: (1) cells cultured on an optically thick polymer substrate measured using an immersion/dipping objective; and (2) cells cultured on a transparent polymer substrate and measured using an inverted optical microscope. In these examples, we show that Raman hyperspectral data sets with sufficient quality can be successfully acquired to map the distribution of common biomolecules in cells, such as nucleic acids, proteins, and lipids, as well as detecting the early stages of apoptosis. We also discuss strategies for further improvements that could expand the application of Raman hyperspectral imaging on polymer substrates even further in biomedical sciences and engineering.

  3. Label-free aptamer biosensor for thrombin detection based on functionalized graphene nanocomposites.

    Science.gov (United States)

    Wang, Qingqing; Zhou, Zhixue; Zhai, Yanling; Zhang, Lingling; Hong, Wei; Zhang, Zhiquan; Dong, Shaojun

    2015-08-15

    A label-free and amplified electrochemical impedimetric aptasensor based on functionalized graphene nanocomposites (rGO-AuNPs) was developed for the detection of thrombin, which played a vital role in thrombosis and hemostasis. The thiolated aptamer and dithiothreitol (TBA15-DTT) were firstly immobilized on the gold electrode to capture the thrombin molecules, and then aptamer functionalized graphene nanocomposites (rGO-TBA29) were used to fabricate a sandwich sensing platform for amplifying the impedimetric signals. As numerous negative charges of TBA29 on the electrode repelled to the [Fe(CN)6](4-/3-) anions, resulting in an obvious amplified charge-transfer resistance (Rct) signal. The Rct increase was linearly proportional to the thrombin concentration from 0.3 to 50nM and a detection limit of 0.01nM thrombin was achieved. In addition, graphene could also be labeled with other probes via electrostatic or π-π stacking interactions to produce signals, therefore different detection methods expanding wide application could be used in this model. Copyright © 2015. Published by Elsevier B.V.

  4. A label-free electrochemical impedance aptasensor for cylindrospermopsin detection based on thionine-graphene nanocomposites.

    Science.gov (United States)

    Zhao, Zhen; Chen, Hongda; Ma, Lina; Liu, Dianjun; Wang, Zhenxin

    2015-08-21

    It is important to develop methods to determine cylindrospermopsin (CYN) at trace levels since CYN is a kind of widespread cyanobacterial toxin in water sources. In this study, a label-free impedimetric aptasensor has been fabricated for detecting CYN. In this case, the amino-substituted aptamer of CYN was covalently grafted onto the surface of the thionine-graphene (TH-G) nanocomposite through the cross-linker glutaraldehyde (GA). The reaction of the aptamer with CYN was monitored by electrochemical impedance spectroscopy because the CYN induced conformation change of the aptamer can cause a remarkable decrease of the electron transfer resistance. Under optimum conditions, the aptasensor exhibits high sensitivity and a low detection limit for CYN determination. The CYN can be quantified in a wide range of 0.39 to 78 ng mL(-1) with a good linearity (R(2) = 0.9968) and a low detection limit of 0.117 ng mL(-1). In addition, the proposed aptasensor displays excellent stability, reusability and reproducibility.

  5. Direct growth of graphene on quartz substrates for label-free detection of adenosine triphosphate

    International Nuclear Information System (INIS)

    Xu, Shicai; Man, Baoyuan; Jiang, Shouzhen; Yue, Weiwei; Yang, Cheng; Liu, Mei; Chen, Chuansong; Zhang, Chao

    2014-01-01

    We demonstrate that continuous, uniform graphene films can be directly synthesized on quartz substrates using a two-temperature-zone chemical vapor deposition system and that their layers can be controlled by adjusting the precursor partial pressure. Raman spectroscopy and transmission electron microscopy confirm the formation of monolayer graphene with a grain size of ∼100 nm. Hall measurements show a room-temperature carrier mobility above 1500 cm 2  V −1  s −1 . The optical transmittance and conductance of the graphene films are comparable to those of transferred metal-catalyzed graphene. The method avoids the complicated and skilled post-growth transfer process and allows the graphene to be directly incorporated into a fully functional biosensor for label-free detection of adenosine triphosphate (ATP). This device shows a fast response time of a few milliseconds and achieves a high sensitivity to ATP molecules over a very wide range from 0.002 to 5 mM. (paper)

  6. Graphene oxide based photoinduced charge transfer label-free near-infrared fluorescent biosensor for dopamine.

    Science.gov (United States)

    Chen, Jin-Long; Yan, Xiu-Ping; Meng, Kang; Wang, Shu-Feng

    2011-11-15

    While the super fluorescence quenching capacity of graphene and graphene oxide (GO) has been extensively employed to develop fluorescent sensors, their own unique fluorescence and its potential for chemo-/biosensing have seldom been explored. Here we report a GO-based photoinduced charge transfer (PCT) label-free near-infrared (near-IR) fluorescent biosensor for dopamine (DA). The multiple noncovalent interactions between GO and DA and the ultrafast decay at the picosecond range of the near-IR fluorescence of GO resulted in effective self-assembly of DA molecules on the surface of GO, and significant fluorescence quenching, allowing development of a PCT-based biosensor with direct readout of the near-IR fluorescence of GO for selective and sensitive detection of DA. The developed method gave a detection limit of 94 nM and a relative standard deviation of 2.0% for 11 replicate detections of 2.0 μM DA and was successfully applied to the determination of DA in biological fluids with quantitative recovery (98-115%).

  7. Graphene-CNT nanohybrid aptasensor for label free detection of cardiac biomarker myoglobin.

    Science.gov (United States)

    Kumar, Vinod; Shorie, Munish; Ganguli, Ashok K; Sabherwal, Priyanka

    2015-10-15

    We report a label free electrochemical detection of cardiac bio-marker myoglobin (Mb) on aptamer functionalized rGO/CNT nanostructured electrodes by measuring its direct electron transfer (DET). Configured as a highly responsive aptasensor, the newly developed biosensing platform exhibits synergistic effect of the nano-hybrid functional construct by combining good electrical properties and the facile chemical functionality of nanohybrid for the compatible bio-interface development. The specific anti-Mb aptamer was generated by five iterative SELEX (Systematic evolution of ligands by exponential enrichment) rounds, showing high senstivity (KD ~65 pM). The aptamer functionalized rGO/CNT nanostructured electrodes demonstrated a significant increase in signal response with a detection limit of ~0.34 ng/mL in the dynamic response range between 1 ng/mL and 4 µg/mL for Mb. The newly developed DET assay format presents a promising candidate in point-of-care diagnosis for routine screening of Mb in patient's samples. Copyright © 2015 Elsevier B.V. All rights reserved.

  8. Enhanced Plasmonic Biosensors of Hybrid Gold Nanoparticle-Graphene Oxide-Based Label-Free Immunoassay

    Science.gov (United States)

    Chiu, Nan-Fu; Chen, Chi-Chu; Yang, Cheng-Du; Kao, Yu-Sheng; Wu, Wei-Ren

    2018-05-01

    In this study, we propose a modified gold nanoparticle-graphene oxide sheet (AuNP-GO) nanocomposite to detect two different interactions between proteins and hybrid nanocomposites for use in biomedical applications. GO sheets have high bioaffinity, which facilitates the attachment of biomolecules to carboxyl groups and has led to its use in the development of sensing mechanisms. When GO sheets are decorated with AuNPs, they introduce localized surface plasmon resonance (LSPR) in the resonance energy transfer of spectral changes. Our results suggest a promising future for AuNP-GO-based label-free immunoassays to detect disease biomarkers and rapidly diagnose infectious diseases. The results showed the detection of antiBSA in 10 ng/ml of hCG non-specific interfering protein with dynamic responses ranging from 1.45 nM to 145 fM, and a LOD of 145 fM. Considering the wide range of potential applications of GO sheets as a host material for a variety of nanoparticles, the approach developed here may be beneficial for the future integration of nanoparticles with GO nanosheets for blood sensing. The excellent anti-interference characteristics allow for the use of the biosensor in clinical analysis and point-of-care testing (POCT) diagnostics of rapid immunoassay products, and it may also be a potential tool for the measurement of biomarkers in human serum.

  9. Low cost label-free live cell imaging for biological samples

    Science.gov (United States)

    Seniya, C.; Towers, C. E.; Towers, D. P.

    2017-02-01

    This paper reports the progress to develop a practical phase measuring microscope offering new capabilities in terms of phase measurement accuracy and quantification of cell:cell interactions over the longer term. A novel, low cost phase interference microscope for imaging live cells (label-free) is described. The method combines the Zernike phase contrast approach with a dual mirror design to enable phase modulation between the scattered and un-scattered optical fields. Two designs are proposed and demonstrated, one of which retains the common path nature of Zernike's original microscopy concept. In both setups the phase shift is simple to control via a piezoelectric driven mirror in the back focal plane of the imaging system. The approach is significantly cheaper to implement than those based on spatial light modulators (SLM) at approximately 20% of the cost. A quantitative assessment of the performance of a set of phase shifting algorithms is also presented, specifically with regard to broad bandwidth illumination in phase contrast microscopy. The simulation results show that the phase measurement accuracy is strongly dependent on the algorithm selected and the optical path difference in the sample.

  10. Impedimetric Label-Free Immunosensor on Disposable Modified Screen-Printed Electrodes for Ochratoxin A

    Directory of Open Access Journals (Sweden)

    Francesca Malvano

    2016-06-01

    Full Text Available An impedimetric label-free immunosensor on disposable screen-printed carbon electrodes (SPCE for quantitative determination of Ochratoxin A (OTA has been developed. After modification of the SPCE surface with gold nanoparticles (AuNPs, the anti-OTA was immobilized on the working electrode through a cysteamine layer. After each coating step, the modified surfaces were characterized by cyclic voltammetry (CV and electrochemical impedance spectroscopy (EIS. The capacitance was chosen as the best parameter that describes the reproducible change in electrical properties of the electrode surface at different OTA concentrations and it was used to investigate the analytical parameters of the developed immunosensor. Under optimized conditions, the immunosensor showed a linear relationship between 0.3 and 20 ng/mL with a low detection limit of 0.25 ng/mL, making it suitable to control OTA content in many common food products. Lastly, the immunosensor was used to measure OTA in red wine samples and the results were compared with those registered with a competitive ELISA kit. The immunosensor was sensitive to OTA lower than 2 μg/kg, which represents the lower acceptable limit of OTA established by European legislation for common food products.

  11. Direct, Label-Free, and Rapid Transistor-Based Immunodetection in Whole Serum.

    Science.gov (United States)

    Gutiérrez-Sanz, Óscar; Andoy, Nesha M; Filipiak, Marcin S; Haustein, Natalie; Tarasov, Alexey

    2017-09-22

    Transistor-based biosensors fulfill many requirements posed upon transducers for future point-of-care diagnostic devices such as scalable fabrication and label-free and real-time quantification of chemical and biological species with high sensitivity. However, the short Debye screening length in physiological samples (<1 nm) has been a major drawback so far, preventing direct measurements in serum. In this work, we demonstrate how tailoring the sensing surface with short specific biological receptors and a polymer polyethylene glycol (PEG) can strongly enhance the sensor response. In addition, the sensor performance can be dramatically improved if the measurements are performed at elevated temperatures (37 °C instead of 21 °C). With this novel approach, highly sensitive and selective detection of a representative immunosensing parameter-human thyroid-stimulating hormone-is shown over a wide measuring range with subpicomolar detection limits in whole serum. To the best of our knowledge, this is the first demonstration of direct immunodetection in whole serum using transistor-based biosensors, without the need for sample pretreatment, labeling, or washing steps. The presented sensor is low-cost, can be easily integrated into portable diagnostics devices, and offers a competitive performance compared to state-of-the-art central laboratory analyzers.

  12. Label-Free Aptasensor for Lysozyme Detection Using Electrochemical Impedance Spectroscopy

    Directory of Open Access Journals (Sweden)

    Dionisia Ortiz-Aguayo

    2018-01-01

    Full Text Available This research develops a label-free aptamer biosensor (aptasensor based on graphite-epoxy composite electrodes (GECs for the detection of lysozyme protein using Electrochemical Impedance Spectroscopy (EIS technique. The chosen immobilization technique was based on covalent bonding using carbodiimide chemistry; for this purpose, carboxylic moieties were first generated on the graphite by electrochemical grafting. The detection was performed using [Fe(CN6]3−/[Fe(CN6]4− as redox probe. After recording the frequency response, values were fitted to its electric model using the principle of equivalent circuits. The aptasensor showed a linear response up to 5 µM for lysozyme and a limit of detection of 1.67 µM. The sensitivity of the established method was 0.090 µM−1 in relative charge transfer resistance values. The interference response by main proteins, such as bovine serum albumin and cytochrome c, has been also characterized. To finally verify the performance of the developed aptasensor, it was applied to wine analysis.

  13. Label-Free Aptasensor for Lysozyme Detection Using Electrochemical Impedance Spectroscopy.

    Science.gov (United States)

    Ortiz-Aguayo, Dionisia; Del Valle, Manel

    2018-01-26

    This research develops a label-free aptamer biosensor (aptasensor) based on graphite-epoxy composite electrodes (GECs) for the detection of lysozyme protein using Electrochemical Impedance Spectroscopy (EIS) technique. The chosen immobilization technique was based on covalent bonding using carbodiimide chemistry; for this purpose, carboxylic moieties were first generated on the graphite by electrochemical grafting. The detection was performed using [Fe(CN)₆] 3- /[Fe(CN)₆] 4- as redox probe. After recording the frequency response, values were fitted to its electric model using the principle of equivalent circuits. The aptasensor showed a linear response up to 5 µM for lysozyme and a limit of detection of 1.67 µM. The sensitivity of the established method was 0.090 µM -1 in relative charge transfer resistance values. The interference response by main proteins, such as bovine serum albumin and cytochrome c, has been also characterized. To finally verify the performance of the developed aptasensor, it was applied to wine analysis.

  14. Development of an enrofloxacin immunosensor based on label-free electrochemical impedance spectroscopy.

    Science.gov (United States)

    Wu, Ching-Chou; Lin, Chia-Hung; Wang, Way-Shyan

    2009-06-30

    Enrofloxacin is the most widespread antibiotic in the fluoroquinolone family. As such, the development of a rapid and sensitive method for the determination of trace amounts of enrofloxacin is an important issue in the health field. The interaction of the enrofloxacin antigen to a specific antibody (Ab) immobilized on an 11-mercapto-undecanoic acid-coated gold electrode was quantified by electrochemical impedance spectroscopy. Two equivalent circuits were separately used to interpret the obtained impedance spectra. These circuits included one resistor in series with one parallel circuit comprised of a resistor and a capacitor (1R//C), and one resistor in series with two parallel RC circuits (2R//C). The results indicate that the antigen-antibody reaction analyzed using the 1R//C circuit provided a more sensitive resistance increment against the enrofloxacin concentration than that of the 2R//C circuit. However, the 2R//C circuit provided a better fitting for impedance spectra, and therefore supplies more detailed results of the enrofloxacin-antibody interaction, causing the increase of electron transfer resistance selectively to the modified layer, and not the electrical double layer. The antibody-modified electrode allowed for analysis of the dynamic linear range of 1-1000 ng/ml enrofloxacin with a detection limit of 1 ng/ml. The reagentless and label-free impedimetric immunosensors provide a simple and sensitive detection method for the specific determination of enrofloxacin.

  15. Silicon photonic resonator for label-free bio-sensing application

    Science.gov (United States)

    Udomsom, Suruk; Mankong, Ukrit; Theera-Umpon, Nipon; Ittipratheep, Nattapol; Umezawa, Toshimasa; Matsumoto, Atsushi; Yamamoto, Naokatsu

    2018-03-01

    In medical diagnostics there is an increasing demand for biosensors that can specifically detect biological analytes in a fluid. Especially label-free sensing, consistings of a transducer with biorecognition molecules immobilized on its surface without relying on fluorescent dye. In this paper we study the design and fabrication of a silicon nanowire photonic ring resonator and its feasibility as a biosensor. We have simulated and fabricated racetrack ring resonators which have a few tenths of micrometer gap, up to 0.5 μm between the input / output waveguides and the resonators. It is found that the devices can be designed with large Q factors. Sensitivity to biomaterial detection has been simulated for antibody (goat anti-mouse IgG) - antigen (mouse IgG) using 3-dimensional Finite Difference Time Domain technique. The simulated results show that the ring resonator has a response 15 nm resonance shift per refractive index unit. Antibody coating method is also discussed in this paper which can be applied to other antibody-antigen types.

  16. Review of Transducer Principles for Label-Free Biomolecular Interaction Analysis

    Directory of Open Access Journals (Sweden)

    Janos Vörös

    2011-07-01

    Full Text Available Label-free biomolecular interaction analysis is an important technique to study the chemical binding between e.g., protein and protein or protein and small molecule in real-time. The parameters obtained with this technique, such as the affinity, are important for drug development. While the surface plasmon resonance (SPR instruments are most widely used, new types of sensors are emerging. These developments are generally driven by the need for higher throughput, lower sample consumption or by the need of complimentary information to the SPR data. This review aims to give an overview about a wide range of sensor transducers, the working principles and the peculiarities of each technology, e.g., concerning the set-up, sensitivity, sensor size or required sample volume. Starting from optical technologies like the SPR and waveguide based sensors, acoustic sensors like the quartz crystal microbalance (QCM and the film bulk acoustic resonator (FBAR, calorimetric and electrochemical sensors are covered. Technologies long established in the market are presented together with those newly commercially available and with technologies in the early development stage. Finally, the commercially available instruments are summarized together with their sensitivity and the number of sensors usable in parallel and an outlook for potential future developments is given.

  17. Label-free, single-object sensing with a microring resonator: FDTD simulation.

    Science.gov (United States)

    Nguyen, Dan T; Norwood, Robert A

    2013-01-14

    Label-free, single-object sensing with a microring resonator is investigated numerically using the finite difference time-domain (FDTD) method. A pulse with ultra-wide bandwidth that spans over several resonant modes of the ring and of the sensing object is used for simulation, enabling a single-shot simulation of the microring sensing. The FDTD simulation not only can describe the circulation of the light in a whispering-gallery-mode (WGM) microring and multiple interactions between the light and the sensing object, but also other important factors of the sensing system, such as scattering and radiation losses. The FDTD results show that the simulation can yield a resonant shift of the WGM cavity modes. Furthermore, it can also extract eigenmodes of the sensing object, and therefore information from deep inside the object. The simulation method is not only suitable for a single object (single molecule, nano-, micro-scale particle) but can be extended to the problem of multiple objects as well.

  18. Label-free silicon photonic biosensor system with integrated detector array

    Science.gov (United States)

    Yan, Rongjin; Mestas, Santano P.; Yuan, Guangwei; Safaisini, Rashid; Dandy, David S.

    2010-01-01

    An integrated, inexpensive, label-free photonic waveguide biosensor system with multi-analyte capability has been implemented on a silicon photonics integrated circuit from a commercial CMOS line and tested with nanofilms. The local evanescent array coupled (LEAC) biosensor is based on a new physical phenomenon that is fundamentally different from the mechanisms of other evanescent field sensors. Increased local refractive index at the waveguide’s upper surface due to the formation of a biological nanofilm causes local modulation of the evanescent field coupled into an array of photodetectors buried under the waveguide. The planar optical waveguide biosensor system exhibits sensitivity of 20%/nm photocurrent modulation in response to adsorbed bovine serum albumin (BSA) layers less than 3 nm thick. In addition to response to BSA, an experiment with patterned photoresist as well as beam propagation method simulations support the evanescent field shift principle. The sensing mechanism enables the integration of all optical and electronic components for a multi-analyte biosensor system on a chip. PMID:19606292

  19. Label-Free Biosensors Based on Bimodal Waveguide (BiMW) Interferometers.

    Science.gov (United States)

    Herranz, Sonia; Gavela, Adrián Fernández; Lechuga, Laura M

    2017-01-01

    The bimodal waveguide (BiMW) sensor is a novel common path interferometric transducer based on the evanescent field detection principle, which in combination with a bio-recognition element allows the direct detection of biomolecular interactions in a label-free scheme. Due to its inherent high sensitivity it has great potential to become a powerful analytical tool for monitoring substances of interest in areas such as environmental control, medical diagnostics and food safety, among others. The BiMW sensor is fabricated using standard silicon-based technology allowing cost-effective production, and meeting the requirements of portability and disposability necessary for implementation in a point-of-care (POC) setting.In this chapter we describe the design and fabrication of the BiMW transducer, as well as its application for bio-sensing purposes. We show as an example the biosensor capabilities two different applications: (1) the immunodetection of Irgarol 1051 biocide useful in the environmental field, and (2) the detection of human growth hormone as used in clinical diagnostics. The detection is performed in real time by monitoring changes in the intensity pattern of light exiting the BiMW transducer resulting from antigen-antibody interactions on the surface of the sensor.

  20. Label-free nanoplasmonic sensing of tumor-associate autoantibodies for early diagnosis of colorectal cancer.

    Science.gov (United States)

    Soler, Maria; Estevez, M-Carmen; Villar-Vazquez, Roi; Casal, J Ignacio; Lechuga, Laura M

    2016-08-03

    Colorectal cancer is treatable and curable when detected at early stages. However there is a lack of less invasive and more specific screening and diagnosis methods which would facilitate its prompt identification. Blood circulating autoantibodies which are immediately produced by the immune system at tumor appearance have become valuable biomarkers for preclinical diagnosis of cancer. In this work, we present the rapid and label-free detection of colorectal cancer autoantibodies directly in blood serum or plasma using a recently developed nanoplasmonic biosensor. Our nanoplasmonic device offers sensitive and real-time quantification of autoantibodies with excellent selectivity and reproducibility, achieving limits of detection around 1 nM (150-160 ng mL(-1)). A preliminary evaluation of clinical samples of colorectal cancer patients has shown good correlation with ELISA. These results demonstrate the reliability of the nanobiosensor strategy and pave the way towards the achievement of a sensitive diagnostic tool for early detection of colorectal cancer. Copyright © 2016 Elsevier B.V. All rights reserved.

  1. Label-free quantitative proteome analysis of the surface-bound salivary pellicle.

    Science.gov (United States)

    Delius, Judith; Trautmann, Simone; Médard, Guillaume; Kuster, Bernhard; Hannig, Matthias; Hofmann, Thomas

    2017-04-01

    The salivary pellicle, covering natural as well as restored tooth surfaces in the oral cavity as an immobilized protein-rich layer, acts as an important physico-chemical and biological mediator at the tooth-saliva-interface. For the first time, the pellicle's proteome of individual volunteers were analyzed separately on three consecutive days and the relative protein abundance determined by a label-free quantitative nano-LC-MS/MS approach. A total of 72 major proteins were identified in the initial pellicles formed intraorally on dental ceramic specimens already after 3min with high inter-individual and inter-day consistency. In comparison, significant differences in protein abundance were evident between subjects, thus indicating unique individual pellicle profiles. Furthermore, the relative protein abundance in pellicles was compared to the proteome pattern in the corresponding saliva samples of the same individuals to provide first data on significantly enriched and depleted salivary proteins (p <0.05) within the surface-bound salivary pellicle. Our findings reveal the initial adsorption of salivary proteins at the solid-liquid interface to be a rapid, highly selective, and reproducible process leading to the immobilization of a broad range of protective proteins and enzymes on the substratum surface within a few minutes. This provides evidence that the pellicle layer might be physiologically functional even without further maturation. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. Magnified Image Spatial Spectrum (MISS) microscopy for nanometer and millisecond scale label-free imaging

    Science.gov (United States)

    Majeed, Hassaan; Ma, Lihong; Lee, Young Jae; Kandel, Mikhail; Min, Eunjung; Jung, Woonggyu; Best-Popescu, Catherine; Popescu, Gabriel

    2018-03-01

    Label-free imaging of rapidly moving, sub-diffraction sized structures has important applications in both biology and material science, as it removes the limitations associated with fluorescence tagging. However, unlabeled nanoscale particles in suspension are difficult to image due to their transparency and fast Brownian motion. Here we describe a novel interferometric imaging technique referred to as Magnified Image Spatial Spectrum (MISS) microscopy, which overcomes these challenges. The MISS microscope provides quantitative phase information and enables dynamic light scattering investigations with an overall optical path length sensitivity of 0.95 nm at 833 frames per second acquisition rate. Using spatiotemporal filtering, we find that the sensitivity can be further pushed down to 0.001-0.01 nm. We demonstrate the instrument's capability through colloidal nanoparticle sizing down to 20 nm diameter and measurements of live neuron membrane dynamics. MISS microscopy is implemented as an upgrade module to an existing microscope, which converts it into a powerful light scattering instrument. Thus, we anticipate that MISS will be adopted broadly for both material and life sciences applications.

  3. Label-free silicon photonic biosensor system with integrated detector array.

    Science.gov (United States)

    Yan, Rongjin; Mestas, Santano P; Yuan, Guangwei; Safaisini, Rashid; Dandy, David S; Lear, Kevin L

    2009-08-07

    An integrated, inexpensive, label-free photonic waveguide biosensor system with multi-analyte capability has been implemented on a silicon photonics integrated circuit from a commercial CMOS line and tested with nanofilms. The local evanescent array coupled (LEAC) biosensor is based on a new physical phenomenon that is fundamentally different from the mechanisms of other evanescent field sensors. Increased local refractive index at the waveguide's upper surface due to the formation of a biological nanofilm causes local modulation of the evanescent field coupled into an array of photodetectors buried under the waveguide. The planar optical waveguide biosensor system exhibits sensitivity of 20%/nm photocurrent modulation in response to adsorbed bovine serum albumin (BSA) layers less than 3 nm thick. In addition to response to BSA, an experiment with patterned photoresist as well as beam propagation method simulations support the evanescent field shift principle. The sensing mechanism enables the integration of all optical and electronic components for a multi-analyte biosensor system on a chip.

  4. Label free quantitative proteomics analysis on the cisplatin resistance in ovarian cancer cells.

    Science.gov (United States)

    Wang, F; Zhu, Y; Fang, S; Li, S; Liu, S

    2017-05-20

    Quantitative proteomics has been made great progress in recent years. Label free quantitative proteomics analysis based on the mass spectrometry is widely used. Using this technique, we determined the differentially expressed proteins in the cisplatin-sensitive ovarian cancer cells COC1 and cisplatin-resistant cells COC1/DDP before and after the application of cisplatin. Using the GO analysis, we classified those proteins into different subgroups bases on their cellular component, biological process, and molecular function. We also used KEGG pathway analysis to determine the key signal pathways that those proteins were involved in. There are 710 differential proteins between COC1 and COC1/DDP cells, 783 between COC1 and COC1/DDP cells treated with cisplatin, 917 between the COC1/DDP cells and COC1/DDP cells treated with LaCl3, 775 between COC1/DDP cells treated with cisplatin and COC1/DDP cells treated with cisplatin and LaCl3. Among the same 411 differentially expressed proteins in cisplatin-sensitive COC1 cells and cisplain-resistant COC1/DDP cells before and after cisplatin treatment, 14% of them were localized on the cell membrane. According to the KEGG results, differentially expressed proteins were classified into 21 groups. The most abundant proteins were involved in spliceosome. This study lays a foundation for deciphering the mechanism for drug resistance in ovarian tumor.

  5. Label-free recognition of drug resistance via impedimetric screening of breast cancer cells.

    Directory of Open Access Journals (Sweden)

    Bilge Eker

    Full Text Available We present a novel study on label-free recognition and distinction of drug resistant breast cancer cells (MCF-7 DOX from their parental cells (MCF-7 WT via impedimetric measurements. Drug resistant cells exhibited significant differences in their dielectric properties compared to wild-type cells, exerting much higher extracellular resistance (Rextra . Immunostaining revealed that MCF-7 DOX cells gained a much denser F-actin network upon acquiring drug resistance indicating that remodeling of actin cytoskeleton is probably the reason behind higher Rextra , providing stronger cell architecture. Moreover, having exposed both cell types to doxorubicin, we were able to distinguish these two phenotypes based on their substantially different drug response. Interestingly, impedimetric measurements identified a concentration-dependent and reversible increase in cell stiffness in the presence of low non-lethal drug doses. Combined with a profound frequency analysis, these findings enabled distinguishing distinct cellular responses during drug exposure within four concentration ranges without using any labeling. Overall, this study highlights the possibility to differentiate drug resistant phenotypes from their parental cells and to assess their drug response by using microelectrodes, offering direct, real-time and noninvasive measurements of cell dependent parameters under drug exposure, hence providing a promising step for personalized medicine applications such as evaluation of the disease progress and optimization of the drug treatment of a patient during chemotherapy.

  6. Label-free investigation of the effects of lithium niobate polarization on cell adhesion

    Science.gov (United States)

    Mandracchia, B.; Gennari, O.; Paturzo, M.; Grilli, S.; Ferraro, P.

    2017-06-01

    The determination of contact area is pivotal to understand how biomaterials properties influence cell adhesion. In particular, the influence of surface charges is well-known but still controversial, especially when new functional materials and methods are introduced. Here, we use for the first time Holographic Total Internal Reflection Microscopy (HoloTIRM) to study the influence of the spontaneous polarization of ferroelectric lithium niobate (LN) on the adhesion properties of fibroblast cells. The selective illumination of a very thin region directly above the substrate, achieved by Total Internal Reflection, provides high-contrast images of the contact regions. Holographic recording, on the other hand, allows for label-free quantitative phase imaging of the contact areas between cells and LN. Phase signal is more sensitive in the first 100nm and, thus more reliable in order to locate focal contacts. This work shows that cells adhering on negatively polarized LN present a significant increase of the contact area in comparison with cells adhering on the positively polarized LN substrate, as well as an intensification of contact vicinity. This confirms the potential of LN as a platform for investigating the role of charges on cellular processes. The similarity of cell adhesion behavior on negatively polarized LN and glass control also confirms the possibility to use LN as an active substrate without impairing cell behavior.

  7. Label-free in vivo optical imaging of functional microcirculations within meninges and cortex in mice.

    Science.gov (United States)

    Jia, Yali; Wang, Ruikang K

    2010-12-15

    Abnormal microcirculation within meninges is common in many neurological diseases. There is a need for an imaging method that is capable of monitoring dynamic meningeal microcirculations, preferably decoupled from cortical blood flow. Optical microangiography (OMAG) is a recently developed label-free imaging method capable of producing 3D images of dynamic blood perfusion within micro-circulatory tissue beds at an imaging depth up to ∼2 mm, with an unprecedented imaging sensitivity to blood flow at ∼4 μm/s. In this paper, we demonstrate the utility of OMAG in imaging the detailed blood flow distributions, at a capillary level resolution, within the meninges and cortex in mice with the cranium left intact. Using a thrombotic mouse model, we show that the OMAG can yield longitudinal measurements of meningeal vascular responses to the insult and can decouple these responses from those in the cortex, giving valuable information regarding the localized hemodynamics along with the dynamic formation of thrombotic event. The results indicate that OMAG can be a useful tool to study therapeutic strategies in preclinical animal models in order to mitigate various pathologies that are mainly related to the meningeal circulations. Copyright © 2010 Elsevier B.V. All rights reserved.

  8. Real-time image processing for label-free enrichment of Actinobacteria cultivated in picolitre droplets.

    Science.gov (United States)

    Zang, Emerson; Brandes, Susanne; Tovar, Miguel; Martin, Karin; Mech, Franziska; Horbert, Peter; Henkel, Thomas; Figge, Marc Thilo; Roth, Martin

    2013-09-21

    The majority of today's antimicrobial therapeutics is derived from secondary metabolites produced by Actinobacteria. While it is generally assumed that less than 1% of Actinobacteria species from soil habitats have been cultivated so far, classic screening approaches fail to supply new substances, often due to limited throughput and frequent rediscovery of already known strains. To overcome these restrictions, we implement high-throughput cultivation of soil-derived Actinobacteria in microfluidic pL-droplets by generating more than 600,000 pure cultures per hour from a spore suspension that can subsequently be incubated for days to weeks. Moreover, we introduce triggered imaging with real-time image-based droplet classification as a novel universal method for pL-droplet sorting. Growth-dependent droplet sorting at frequencies above 100 Hz is performed for label-free enrichment and extraction of microcultures. The combination of both cultivation of Actinobacteria in pL-droplets and real-time detection of growing Actinobacteria has great potential in screening for yet unknown species as well as their undiscovered natural products.

  9. Rapid and label-free detection of protein a by aptamer-tethered porous silicon nanostructures.

    Science.gov (United States)

    Urmann, Katharina; Reich, Peggy; Walter, Johanna-Gabriela; Beckmann, Dieter; Segal, Ester; Scheper, Thomas

    2017-09-10

    Protein A, which is secreted by and displayed on the cell membrane of Staphylococcus aureus is an important biomarker for S. aureus. Thus, its rapid and specific detection may facilitate the pathogen identification and initiation of proper treatment. Herein, we present a simple, label-free and rapid optical biosensor enabling specific detection of protein A. Protein A-binding aptamer serves as the capture probe and is immobilized onto a nanostructured porous silicon thin film, which serves as the optical transducer element. We demonstrate high sensitivity of the biosensor with a linear detection range between 8 and 23μM. The apparent dissociation constant was determined as 13.98μM and the LoD is 3.17μM. Harnessing the affinity between protein A and antibodies, a sandwich assay format was developed to amplify the optical signal associated with protein A capture by the aptamer. Using this approach, we increase the sensitivity of the biosensor, resulting in a three times lower LoD. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. Targeted radiosensitization of cells expressing truncated DNA polymerase {beta}.

    NARCIS (Netherlands)

    Neijenhuis, S.; Verwijs-Janssen, M.; Broek, Bart van den; Begg, A.C.; Vens, C.

    2010-01-01

    Ionizing radiation (IR) is an effective anticancer treatment, although failures still occur. To improve radiotherapy, tumor-targeted strategies are needed to increase radiosensitivity of tumor cells, without influencing normal tissue radiosensitivity. Base excision repair (BER) and single-strand

  11. Mung bean nuclease treatment increases capture specificity of microdroplet-PCR based targeted DNA enrichment.

    Directory of Open Access Journals (Sweden)

    Zhenming Yu

    Full Text Available Targeted DNA enrichment coupled with next generation sequencing has been increasingly used for interrogation of select sub-genomic regions at high depth of coverage in a cost effective manner. Specificity measured by on-target efficiency is a key performance metric for target enrichment. Non-specific capture leads to off-target reads, resulting in waste of sequencing throughput on irrelevant regions. Microdroplet-PCR allows simultaneous amplification of up to thousands of regions in the genome and is among the most commonly used strategies for target enrichment. Here we show that carryover of single-stranded template genomic DNA from microdroplet-PCR constitutes a major contributing factor for off-target reads in the resultant libraries. Moreover, treatment of microdroplet-PCR enrichment products with a nuclease specific to single-stranded DNA alleviates off-target load and improves enrichment specificity. We propose that nuclease treatment of enrichment products should be incorporated in the workflow of targeted sequencing using microdroplet-PCR for target capture. These findings may have a broad impact on other PCR based applications for which removal of template DNA is beneficial.

  12. A type III-B CRISPR-Cas effector complex mediating massive target DNA destruction.

    Science.gov (United States)

    Han, Wenyuan; Li, Yingjun; Deng, Ling; Feng, Mingxia; Peng, Wenfang; Hallstrøm, Søren; Zhang, Jing; Peng, Nan; Liang, Yun Xiang; White, Malcolm F; She, Qunxin

    2017-02-28

    The CRISPR (clustered regularly interspaced short palindromic repeats) system protects archaea and bacteria by eliminating nucleic acid invaders in a crRNA-guided manner. The Sulfolobus islandicus type III-B Cmr-α system targets invading nucleic acid at both RNA and DNA levels and DNA targeting relies on the directional transcription of the protospacer in vivo. To gain further insight into the involved mechanism, we purified a native effector complex of III-B Cmr-α from S. islandicus and characterized it in vitro. Cmr-α cleaved RNAs complementary to crRNA present in the complex and its ssDNA destruction activity was activated by target RNA. The ssDNA cleavage required mismatches between the 5΄-tag of crRNA and the 3΄-flanking region of target RNA. An invader plasmid assay showed that mutation either in the histidine-aspartate acid (HD) domain (a quadruple mutation) or in the GGDD motif of the Cmr-2α protein resulted in attenuation of the DNA interference in vivo. However, double mutation of the HD motif only abolished the DNase activity in vitro. Furthermore, the activated Cmr-α binary complex functioned as a highly active DNase to destroy a large excess DNA substrate, which could provide a powerful means to rapidly degrade replicating viral DNA. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  13. Mitochondrial Targeted Endonuclease III DNA Repair Enzyme Protects against Ventilator Induced Lung Injury in Mice

    Directory of Open Access Journals (Sweden)

    Masahiro Hashizume

    2014-08-01

    Full Text Available The mitochondrial targeted DNA repair enzyme, 8-oxoguanine DNA glycosylase 1, was previously reported to protect against mitochondrial DNA (mtDNA damage and ventilator induced lung injury (VILI. In the present study we determined whether mitochondrial targeted endonuclease III (EndoIII which cleaves oxidized pyrimidines rather than purines from damaged DNA would also protect the lung. Minimal injury from 1 h ventilation at 40 cmH2O peak inflation pressure (PIP was reversed by EndoIII pretreatment. Moderate lung injury due to ventilation for 2 h at 40 cmH2O PIP produced a 25-fold increase in total extravascular albumin space, a 60% increase in W/D weight ratio, and marked increases in MIP-2 and IL-6. Oxidative mtDNA damage and decreases in the total tissue glutathione (GSH and the GSH/GSSH ratio also occurred. All of these indices of injury were attenuated by mitochondrial targeted EndoIII. Massive lung injury caused by 2 h ventilation at 50 cmH2O PIP was not attenuated by EndoIII pretreatment, but all untreated mice died prior to completing the two hour ventilation protocol, whereas all EndoIII-treated mice lived for the duration of ventilation. Thus, mitochondrial targeted DNA repair enzymes were protective against mild and moderate lung damage and they enhanced survival in the most severely injured group.

  14. TAL effectors: highly adaptable phytobacterial virulence factors and readily engineered DNA targeting proteins

    Science.gov (United States)

    Doyle, Erin L.; Stoddard, Barry L.; Voytas, Daniel F.; Bogdanove, Adam J.

    2013-01-01

    Transcription activator-like (TAL) effectors are transcription factors injected into plant cells by pathogenic bacteria in the genus Xanthomonas. They function as virulence factors by activating host genes important for disease, or as avirulence factors by turning on genes that provide resistance. DNA binding specificity is encoded by polymorphic repeats in each protein that correspond one-to-one with different nucleotides. This code has facilitated target identification and opened new avenues for engineering disease resistance. It has also enabled TAL effector customization for targeted gene control, genome editing, and other applications. This article reviews the structural basis for TAL effector-DNA specificity, the impact of the TAL effector-DNA code on plant pathology and engineered resistance, and recent accomplishments and future challenges in TAL effector-based DNA targeting. PMID:23707478

  15. Targeting and tracing of specific DNA sequences with dTALEs in living cells

    Science.gov (United States)

    Thanisch, Katharina; Schneider, Katrin; Morbitzer, Robert; Solovei, Irina; Lahaye, Thomas; Bultmann, Sebastian; Leonhardt, Heinrich

    2014-01-01

    Epigenetic regulation of gene expression involves, besides DNA and histone modifications, the relative positioning of DNA sequences within the nucleus. To trace specific DNA sequences in living cells, we used programmable sequence-specific DNA binding of designer transcription activator-like effectors (dTALEs). We designed a recombinant dTALE (msTALE) with variable repeat domains to specifically bind a 19-bp target sequence of major satellite DNA. The msTALE was fused with green fluorescent protein (GFP) and stably expressed in mouse embryonic stem cells. Hybridization with a major satellite probe (3D-fluorescent in situ hybridization) and co-staining for known cellular structures confirmed in vivo binding of the GFP-msTALE to major satellite DNA present at nuclear chromocenters. Dual tracing of major satellite DNA and the replication machinery throughout S-phase showed co-localization during mid to late S-phase, directly demonstrating the late replication timing of major satellite DNA. Fluorescence bleaching experiments indicated a relatively stable but still dynamic binding, with mean residence times in the range of minutes. Fluorescently labeled dTALEs open new perspectives to target and trace DNA sequences and to monitor dynamic changes in subnuclear positioning as well as interactions with functional nuclear structures during cell cycle progression and cellular differentiation. PMID:24371265

  16. Targeting and tracing of specific DNA sequences with dTALEs in living cells.

    Science.gov (United States)

    Thanisch, Katharina; Schneider, Katrin; Morbitzer, Robert; Solovei, Irina; Lahaye, Thomas; Bultmann, Sebastian; Leonhardt, Heinrich

    2014-04-01

    Epigenetic regulation of gene expression involves, besides DNA and histone modifications, the relative positioning of DNA sequences within the nucleus. To trace specific DNA sequences in living cells, we used programmable sequence-specific DNA binding of designer transcription activator-like effectors (dTALEs). We designed a recombinant dTALE (msTALE) with variable repeat domains to specifically bind a 19-bp target sequence of major satellite DNA. The msTALE was fused with green fluorescent protein (GFP) and stably expressed in mouse embryonic stem cells. Hybridization with a major satellite probe (3D-fluorescent in situ hybridization) and co-staining for known cellular structures confirmed in vivo binding of the GFP-msTALE to major satellite DNA present at nuclear chromocenters. Dual tracing of major satellite DNA and the replication machinery throughout S-phase showed co-localization during mid to late S-phase, directly demonstrating the late replication timing of major satellite DNA. Fluorescence bleaching experiments indicated a relatively stable but still dynamic binding, with mean residence times in the range of minutes. Fluorescently labeled dTALEs open new perspectives to target and trace DNA sequences and to monitor dynamic changes in subnuclear positioning as well as interactions with functional nuclear structures during cell cycle progression and cellular differentiation.

  17. An Adenovirus DNA Replication Factor, but Not Incoming Genome Complexes, Targets PML Nuclear Bodies.

    Science.gov (United States)

    Komatsu, Tetsuro; Nagata, Kyosuke; Wodrich, Harald

    2016-02-01

    Promyelocytic leukemia protein nuclear bodies (PML-NBs) are subnuclear domains implicated in cellular antiviral responses. Despite the antiviral activity, several nuclear replicating DNA viruses use the domains as deposition sites for the incoming viral genomes and/or as sites for viral DNA replication, suggesting that PML-NBs are functionally relevant during early viral infection to establish productive replication. Although PML-NBs and their components have also been implicated in the adenoviral life cycle, it remains unclear whether incoming adenoviral genome complexes target PML-NBs. Here we show using immunofluorescence and live-cell imaging analyses that incoming adenovirus genome complexes neither localize at nor recruit components of PML-NBs during early phases of infection. We further show that the viral DNA binding protein (DBP), an early expressed viral gene and essential DNA replication factor, independently targets PML-NBs. We show that DBP oligomerization is required to selectively recruit the PML-NB components Sp100 and USP7. Depletion experiments suggest that the absence of one PML-NB component might not affect the recruitment of other components toward DBP oligomers. Thus, our findings suggest a model in which an adenoviral DNA replication factor, but not incoming viral genome complexes, targets and modulates PML-NBs to support a conducive state for viral DNA replication and argue against a generalized concept that PML-NBs target incoming viral genomes. The immediate fate upon nuclear delivery of genomes of incoming DNA viruses is largely unclear. Early reports suggested that incoming genomes of herpesviruses are targeted and repressed by PML-NBs immediately upon nuclear import. Genome localization and/or viral DNA replication has also been observed at PML-NBs for other DNA viruses. Thus, it was suggested that PML-NBs may immediately sense and target nuclear viral genomes and hence serve as sites for deposition of incoming viral genomes and

  18. Sequence-selective targeting of duplex DNA by peptide nucleic acids

    DEFF Research Database (Denmark)

    Nielsen, Peter E

    2010-01-01

    Sequence-selective gene targeting constitutes an attractive drug-discovery approach for genetic therapy, with the aim of reducing or enhancing the activity of specific genes at the transcriptional level, or as part of a methodology for targeted gene repair. The pseudopeptide DNA mimic peptide...

  19. Sci—Fri AM: Mountain — 04: Label-free Raman spectroscopy of single tumour cells detects early radiation-induced glycogen synthesis associated with increased radiation resistance

    International Nuclear Information System (INIS)

    Matthews, Q; Lum, JJ; Isabelle, M; Harder, S; Jirasek, A; Brolo, AG

    2014-01-01

    Purpose: To use label-free Raman spectroscopy (RS) for early treatment monitoring of tumour cell radioresistance. Methods: Three human tumour cell lines, two radioresistant (H460, SF 2 = 0.57 and MCF7, SF 2 = 0.70) and one radiosensitive (LNCaP, SF 2 = 0.36), were irradiated with single fractions of 2, 4, 6, 8 or 10 Gy. In additional experiments, H460 and MCF7 cells were irradiated under co-treatment with the anti-diabetic drug metformin, a known radiosensitizing agent. Treated and control cultures were analyzed with RS daily for 3 days post-treatment. Single-cell Raman spectra were acquired from 20 live cells per sample, and experiments were repeated in triplicate. The combined data sets were analyzed with principal component analysis using standard algorithms. Cells from each culture were also subjected to standard assays for viability, proliferation, cell cycle, and radiation clonogenic survival. Results: The radioresistant cells (H460, MCF7) exhibited a RS molecular radiation response signature, detectable as early as 1 day post-treatment, of which radiation-induced glycogen synthesis is a significant contributor. The radiosensitive cells (LNCaP) exhibited negligible glycogen synthesis. Co-treatment with metformin in MCF7 cells blocked glycogen synthesis, reduced viability and proliferation, and increased radiosensitivity. Conversely, metformin co-treatment in H460 cells did not produce these same effects; importantly, both radiation-induced synthesis of glycogen and radiosensitivity were unaffected. Conclusions: Label-free RS can detect early glycogen synthesis post-irradiation, a previously undocumented metabolic mechanism associated with tumour cell radioresistance that can be targeted to increase radiosensitivity. RS monitoring of intratumoral glycogen may provide new opportunities for personalized combined modality radiotherapy treatments

  20. Gel-free/label-free proteomic, photosynthetic, and biochemical analysis of cowpea (Vigna unguiculata [L.] Walp.) resistance against Cowpea severe mosaic virus (CPSMV).

    Science.gov (United States)

    Varela, Anna Lidia N; Komatsu, Setsuko; Wang, Xin; Silva, Rodolpho G G; Souza, Pedro Filho N; Lobo, Ana Karla M; Vasconcelos, Ilka M; Silveira, Joaquim A G; Oliveira, Jose T A

    2017-06-23

    Cowpea severe mosaic virus (CPSMV) causes significant losses in cowpea (Vigna unguiculata) production. In this present study biochemical, physiological, and proteomic analysis were done to identify pathways and defense proteins that are altered during the incompatible interaction between the cowpea genotype BRS-Marataoã and CPSMV. The leaf protein extracts from mock- (MI) and CPSMV-inoculated plantlets (V) were evaluated at 2 and 6days post-inoculation (DPI). Data support the assumptions that increases in biochemical (high hydrogen peroxide, antioxidant enzymes, and secondary compounds) and physiological responses (high photosynthesis index and chlorophyll content), confirmed by label-free comparative proteomic approach, in which quantitative changes in proteasome proteins, proteins related to photosynthesis, redox homeostasis, regulation factors/RNA processing proteins were observed may be implicated in the resistance of BRS-Marataoã to CPSMV. This pioneering study provides information for the selection of specific pathways and proteins, altered in this incompatible relationship, which could be chosen as targets for detailed studies to advance our understanding of the molecular, physiological, and biochemistry basis of the resistance mechanism of cowpea and design approachs to engineer plants that are more productive. This is a pioneering study in which an incompatible relationship between a resistant cowpea and Cowpea severe mosaic virus (CPSMV) was conducted to comparatively evaluate proteomic profiles by Gel-free/label-free methodology and some physiological and biochemical parameters to shed light on how a resistant cowpea cultivar deals with the virus attack. Specific proteins and associated pathways were altered in the cowpea plants challenged with CPSMV and will contribute to our knowledge on the biological process tailored by cowpea in response to CPSMV. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. Sci—Fri AM: Mountain — 04: Label-free Raman spectroscopy of single tumour cells detects early radiation-induced glycogen synthesis associated with increased radiation resistance

    Energy Technology Data Exchange (ETDEWEB)

    Matthews, Q; Lum, JJ [BC Cancer Agency — Vancouver Island Centre (Canada); Isabelle, M; Harder, S; Jirasek, A [Physics and Astronomy, University of Victoria (Australia); Brolo, AG [Chemistry, University of Victoria (Australia)

    2014-08-15

    Purpose: To use label-free Raman spectroscopy (RS) for early treatment monitoring of tumour cell radioresistance. Methods: Three human tumour cell lines, two radioresistant (H460, SF{sub 2} = 0.57 and MCF7, SF{sub 2} = 0.70) and one radiosensitive (LNCaP, SF{sub 2} = 0.36), were irradiated with single fractions of 2, 4, 6, 8 or 10 Gy. In additional experiments, H460 and MCF7 cells were irradiated under co-treatment with the anti-diabetic drug metformin, a known radiosensitizing agent. Treated and control cultures were analyzed with RS daily for 3 days post-treatment. Single-cell Raman spectra were acquired from 20 live cells per sample, and experiments were repeated in triplicate. The combined data sets were analyzed with principal component analysis using standard algorithms. Cells from each culture were also subjected to standard assays for viability, proliferation, cell cycle, and radiation clonogenic survival. Results: The radioresistant cells (H460, MCF7) exhibited a RS molecular radiation response signature, detectable as early as 1 day post-treatment, of which radiation-induced glycogen synthesis is a significant contributor. The radiosensitive cells (LNCaP) exhibited negligible glycogen synthesis. Co-treatment with metformin in MCF7 cells blocked glycogen synthesis, reduced viability and proliferation, and increased radiosensitivity. Conversely, metformin co-treatment in H460 cells did not produce these same effects; importantly, both radiation-induced synthesis of glycogen and radiosensitivity were unaffected. Conclusions: Label-free RS can detect early glycogen synthesis post-irradiation, a previously undocumented metabolic mechanism associated with tumour cell radioresistance that can be targeted to increase radiosensitivity. RS monitoring of intratumoral glycogen may provide new opportunities for personalized combined modality radiotherapy treatments.

  2. Mitochondrial DNA is a direct target of anti-cancer anthracycline drugs

    International Nuclear Information System (INIS)

    Ashley, Neil; Poulton, Joanna

    2009-01-01

    The anthracyclines, such as doxorubicin (DXR), are potent anti-cancer drugs but they are limited by their clinical toxicity. The mechanisms involved remain poorly understood partly because of the difficulty in determining sub-cellular drug localisation. Using a novel method utilising the fluorescent DNA dye PicoGreen, we found that anthracyclines intercalated not only into nuclear DNA but also mitochondrial DNA (mtDNA). Intercalation of mtDNA by anthracyclines may thus contribute to the marked mitochondrial toxicity associated with these drugs. By contrast, ethidium bromide intercalated exclusively into mtDNA, without interacting with nuclear DNA, thereby explaining why mtDNA is the main target for ethidium. By exploiting PicoGreen quenching we also developed a novel assay for quantification of mtDNA levels by flow-cytometry, an approach which should be useful for studies of mitochondrial dysfunction. In summary our PicoGreen assay should be useful to study drug/DNA interactions within live cells, and facilitate therapeutic drug monitoring and kinetic studies in cancer patients.

  3. A label free aptasensor for Ochratoxin A detection in cocoa beans: An application to chocolate industries

    International Nuclear Information System (INIS)

    Mishra, Rupesh K.; Hayat, Akhtar; Catanante, Gaëlle; Ocaña, Cristina; Marty, Jean-Louis

    2015-01-01

    Contamination of food by mycotoxin occurs in minute/trace quantities. Nearly 92.5% of the cocoa samples present Ochratoxin A (OTA) levels at trace quantity. Hence, there is a necessity for a highly sensitive and selective device that can detect and quantify these organic toxins in various matrices such as cocoa beans. This work reports for the first time, a facile and label-free electrochemical impedimetric aptasensor for rapid detection and quantitation of OTA in cocoa beans. The developed aptasensor was constructed based on the diazonium-coupling reaction mechanism for the immobilization of anti-OTA-aptamer on screen printed carbon electrodes (SPCEs). The aptasensor exhibited a very good limit of detection (LOD) as low as 0.15 ng/mL, with added advantages of good selectivity and reproducibility. The increase in electron transfer resistance was linearly proportional to the OTA concentration in the range 0.15–2.5 ng/mL, with an acceptable recovery percentage (91–95%, RSD = 4.8%) obtained in cocoa samples. This work can facilitate a general model for the detection of OTA in cocoa beans based on the impedimetric aptasensor. The analysis can be performed onsite with pre-constructed and aptamer modified electrodes employing a portable EIS set up. - Highlights: • Simple and facile method to detect OTA. • The aptasensor exhibited a very good limit of detection (LOD) as low as 0.15 ng/mL. • The first report on OTA detection in cocoa beans using impedimetric aptasensor

  4. A label free aptasensor for Ochratoxin A detection in cocoa beans: An application to chocolate industries

    Energy Technology Data Exchange (ETDEWEB)

    Mishra, Rupesh K. [IMAGES, Université De Perpignan Via Domitia, 52 Avenue Paul Alduy, Perpignan Cedex 66860 (France); Hayat, Akhtar [IMAGES, Université De Perpignan Via Domitia, 52 Avenue Paul Alduy, Perpignan Cedex 66860 (France); Interdisciplinary Research Centre in Biomedical Materials (IRCBM), COMSATS Institute of Information Technology (CIIT), Lahore 54000 (Pakistan); Catanante, Gaëlle; Ocaña, Cristina [IMAGES, Université De Perpignan Via Domitia, 52 Avenue Paul Alduy, Perpignan Cedex 66860 (France); Marty, Jean-Louis, E-mail: jlmarty@univ-perp.fr [IMAGES, Université De Perpignan Via Domitia, 52 Avenue Paul Alduy, Perpignan Cedex 66860 (France)

    2015-08-19

    Contamination of food by mycotoxin occurs in minute/trace quantities. Nearly 92.5% of the cocoa samples present Ochratoxin A (OTA) levels at trace quantity. Hence, there is a necessity for a highly sensitive and selective device that can detect and quantify these organic toxins in various matrices such as cocoa beans. This work reports for the first time, a facile and label-free electrochemical impedimetric aptasensor for rapid detection and quantitation of OTA in cocoa beans. The developed aptasensor was constructed based on the diazonium-coupling reaction mechanism for the immobilization of anti-OTA-aptamer on screen printed carbon electrodes (SPCEs). The aptasensor exhibited a very good limit of detection (LOD) as low as 0.15 ng/mL, with added advantages of good selectivity and reproducibility. The increase in electron transfer resistance was linearly proportional to the OTA concentration in the range 0.15–2.5 ng/mL, with an acceptable recovery percentage (91–95%, RSD = 4.8%) obtained in cocoa samples. This work can facilitate a general model for the detection of OTA in cocoa beans based on the impedimetric aptasensor. The analysis can be performed onsite with pre-constructed and aptamer modified electrodes employing a portable EIS set up. - Highlights: • Simple and facile method to detect OTA. • The aptasensor exhibited a very good limit of detection (LOD) as low as 0.15 ng/mL. • The first report on OTA detection in cocoa beans using impedimetric aptasensor.

  5. A label-free silicon quantum dots-based photoluminescence sensor for ultrasensitive detection of pesticides.

    Science.gov (United States)

    Yi, Yinhui; Zhu, Gangbing; Liu, Chang; Huang, Yan; Zhang, Youyu; Li, Haitao; Zhao, Jiangna; Yao, Shouzhuo

    2013-12-03

    Sensitive, rapid, and simple detection methods for the screening of extensively used organophosphorus pesticides and highly toxic nerve agents are in urgent demand. A novel label-free silicon quantum dots (SiQDs)-based sensor was designed for ultrasensitive detection of pesticides. This sensing strategy involves the reaction of acetylcholine chloride (ACh) with acetylcholinesterase (AChE) to form choline that is in turn catalytically oxidized by choline oxidase (ChOx) to produce betaine and H2O2 which can quench the photoluminescence (PL) of SiQDs. Upon the addition of pesticides, the activity of AChE is inhibited, leading to the decrease of the generated H2O2, and hence the PL of SiQDs increases. By measuring the increase in SiQDs PL, the inhibition efficiency of pesticide to AChE activity was evaluated. It was found that the inhibition efficiency was linearly dependent on the logarithm of the pesticides concentration. Consequently, pesticides, such as carbaryl, parathion, diazinon, and phorate, were determined with the SiQDs PL sensing method. The lowest detectable concentrations for carbaryl, parathion, diazinon, and phorate reached 7.25 × 10(-9), 3.25 × 10(-8), 6.76 × 10(-8), and 1.9 × 10(-7) g/L, respectively, which were much lower than those previously reported. The detecting results of pesticide residues in food samples via this method agree well with those from high-performance liquid chromatography. The simple strategy reported here should be suitable for on-site pesticides detection, especially in combination with other portable platforms.

  6. Label-free bimodal waveguide immunosensor for rapid diagnosis of bacterial infections in cirrhotic patients.

    Science.gov (United States)

    Maldonado, Jesús; González-Guerrero, Ana Belén; Domínguez, Carlos; Lechuga, Laura M

    2016-11-15

    Spontaneous bacterial peritonitis is an acute bacterial infection of ascitic fluid; it has a high incidence in cirrhotic patients and it is associated with high mortality. In such a situation, early diagnosis and treatment is crucial for the survival of the patient. However, bacterial analysis in ascitic fluid is currently based on culture methods, which are time-consuming and laborious. We report here the application of a photonic interferometer biosensor based on a bimodal waveguide (BiMW) for the rapid and label-free detection of bacteria directly in ascitic fluid. The device consists of a straight waveguide in which two modes of the same polarization interfere while interacting with the external medium through their evanescent fields. A bimolecular event occurring on the sensor area of the device (e.g. capturing bacteria) will differently affect each light mode, inducing a variation in the phase of the light exiting at the output of the waveguide. In this work, we demonstrate the quantitative detection of Bacillus cereus in buffer medium and Escherichia coli in undiluted ascitic fluid from cirrhotic patients. In the case of Bacillus cereus detection, the device was able to specifically detect bacteria at relevant concentrations in 12.5min and in the case of Escherichia coli detection, the analysis time was 25min. Extrapolation of the data demonstrated that the detection limits of the biosensor could reach few bacteria per milliliter. Based on the results obtained, we consider that the BiMW biosensor is positioned as a promising new clinical tool for user-friendly, cost-effective and real-time microbiological analysis. Copyright © 2016 Elsevier B.V. All rights reserved.

  7. In vitro Selection and Interaction Studies of a DNA Aptamer Targeting Protein A

    OpenAIRE

    Stoltenburg, Regina; Schubert, Thomas; Strehlitz, Beate

    2015-01-01

    A new DNA aptamer targeting Protein A is presented. The aptamer was selected by use of the FluMag-SELEX procedure. The SELEX technology (Systematic Evolution of Ligands by EXponential enrichment) is widely applied as an in vitro selection and amplification method to generate target-specific aptamers and exists in various modified variants. FluMag-SELEX is one of them and is characterized by the use of magnetic beads for target immobilization and fluorescently labeled oligonucleotides for moni...

  8. Inhibition of DNA2 nuclease as a therapeutic strategy targeting replication stress in cancer cells.

    Science.gov (United States)

    Kumar, S; Peng, X; Daley, J; Yang, L; Shen, J; Nguyen, N; Bae, G; Niu, H; Peng, Y; Hsieh, H-J; Wang, L; Rao, C; Stephan, C C; Sung, P; Ira, G; Peng, G

    2017-04-17

    Replication stress is a characteristic feature of cancer cells, which is resulted from sustained proliferative signaling induced by activation of oncogenes or loss of tumor suppressors. In cancer cells, oncogene-induced replication stress manifests as replication-associated lesions, predominantly double-strand DNA breaks (DSBs). An essential mechanism utilized by cells to repair replication-associated DSBs is homologous recombination (HR). In order to overcome replication stress and survive, cancer cells often require enhanced HR repair capacity. Therefore, the key link between HR repair and cellular tolerance to replication-associated DSBs provides us with a mechanistic rationale for exploiting synthetic lethality between HR repair inhibition and replication stress. DNA2 nuclease is an evolutionarily conserved essential enzyme in replication and HR repair. Here we demonstrate that DNA2 is overexpressed in pancreatic cancers, one of the deadliest and more aggressive forms of human cancers, where mutations in the KRAS are present in 90-95% of cases. In addition, depletion of DNA2 significantly reduces pancreatic cancer cell survival and xenograft tumor growth, suggesting the therapeutic potential of DNA2 inhibition. Finally, we develop a robust high-throughput biochemistry assay to screen for inhibitors of the DNA2 nuclease activity. The top inhibitors were shown to be efficacious against both yeast Dna2 and human DNA2. Treatment of cancer cells with DNA2 inhibitors recapitulates phenotypes observed upon DNA2 depletion, including decreased DNA double strand break end resection and attenuation of HR repair. Similar to genetic ablation of DNA2, chemical inhibition of DNA2 selectively attenuates the growth of various cancer cells with oncogene-induced replication stress. Taken together, our findings open a new avenue to develop a new class of anticancer drugs by targeting druggable nuclease DNA2. We propose DNA2 inhibition as new strategy in cancer therapy by targeting

  9. A universal molecular translator for non-nucleic acid targets that enables dynamic DNA assemblies and logic operations.

    Science.gov (United States)

    Tang, Wei; Hu, Shichao; Wang, Huaming; Zhao, Yan; Li, Na; Liu, Feng

    2014-11-28

    A universal molecular translator based on the target-triggered DNA strand displacement was developed, which was able to convert various kinds of non-nucleic acid targets into a unique output DNA. This translation strategy was successfully applied in directing dynamic DNA assemblies and in realizing three-input logic gate operations.

  10. Conducting electrospun fibres with polyanionic grafts as highly selective, label-free, electrochemical biosensor with a low detection limit for non-Hodgkin lymphoma gene.

    Science.gov (United States)

    Kerr-Phillips, Thomas E; Aydemir, Nihan; Chan, Eddie Wai Chi; Barker, David; Malmström, Jenny; Plesse, Cedric; Travas-Sejdic, Jadranka

    2018-02-15

    A highly selective, label-free sensor for the non-Hodgkin lymphoma gene, with an aM detection limit, utilizing electrochemical impedance spectroscopy (EIS) is presented. The sensor consists of a conducting electrospun fibre mat, surface-grafted with poly(acrylic acid) (PAA) brushes and a conducting polymer sensing element with covalently attached oligonucleotide probes. The sensor was fabricated from electrospun NBR rubber, embedded with poly(3,4-ethylenedioxythiophene) (PEDOT), followed by grafting poly(acrylic acid) brushes and then electrochemically polymerizing a conducting polymer monomer with ssDNA probe sequence pre-attached. The resulting non-Hodgkin lymphoma gene sensor showed a detection limit of 1aM (1 × 10 -18 mol/L), more than 400 folds lower compared to a thin-film analogue. The sensor presented extraordinary selectivity, with only 1%, 2.7% and 4.6% of the signal recorded for the fully non-complimentary, T-A and G-C base mismatch oligonucleotide sequences, respectively. We suggest that such greatly enhanced selectivity is due to the presence of negatively charged carboxylic acid moieties from PAA grafts that electrostatically repel the non-complementary and mismatch DNA sequences, overcoming the non-specific binding. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. Inactivation of Pol θ and C-NHEJ eliminates off-target integration of exogenous DNA.

    Science.gov (United States)

    Zelensky, Alex N; Schimmel, Joost; Kool, Hanneke; Kanaar, Roland; Tijsterman, Marcel

    2017-07-07

    Off-target or random integration of exogenous DNA hampers precise genomic engineering and presents a safety risk in clinical gene therapy strategies. Genetic definition of random integration has been lacking for decades. Here, we show that the A-family DNA polymerase θ (Pol θ) promotes random integration, while canonical non-homologous DNA end joining plays a secondary role; cells double deficient for polymerase θ and canonical non-homologous DNA end joining are devoid of any integration events, demonstrating that these two mechanisms define random integration. In contrast, homologous recombination is not reduced in these cells and gene targeting is improved to 100% efficiency. Such complete reversal of integration outcome, from predominately random integration to exclusively gene targeting, provides a rational way forward to improve the efficacy and safety of DNA delivery and gene correction approaches.Random off-target integration events can impair precise gene targeting and poses a safety risk for gene therapy. Here the authors show that repression of polymerase θ and classical non-homologous recombination eliminates random integration.

  12. Dendritic cell targeted liposomes–protamine–DNA complexes mediated by synthetic mannosylated cholestrol as a potential carrier for DNA vaccine

    International Nuclear Information System (INIS)

    Li Pan; Chen Simu; Jiang Yuhong; Jiang Jiayu; Zhang Zhirong; Sun Xun

    2013-01-01

    To construct mannosylated liposomes/protamine/DNA (LPD) carriers for DNA vaccine targeting to dendritic cells (DCs), a mannosylated cholesterol derivative (Man-C6-Chol) was synthesized via simple ester linkage and amide bonds. Then, the Man-C6-Chol was applied to LPD formulation as a synthetic ligand. The physicochemical properties of mannosylated LPD (Man-LPD) were first evaluated, including the size and zeta potential, morphology and the ability to protect DNA against DNase I degradation. Man-LPD showed a small size with a stable viral-like structure. In comparison to non-mannose liposomes/LPD (Man-free liposomes/LPD), mannosylated liposomes/LPD (Man-liposomes/Man-LPD) exhibited higher efficiency in both intracellular uptake (2.3-fold) and transfection (4.5-fold) in vitro. Subsequent MTT assays indicated that the LPD carriers had low toxicity on the tested cells. Afterwards, the investigation into the maturation activation on primary bone marrow-derived DCs (BMDCs) showed that both Man-LPD and Man-free LPD induced remarkable up-regulation of CD80, CD86 and CD40 on BMDCs. Inspired by these studies, we can conclude that the synthetic mannosylated LPD targeting to DCs was a potential carrier for DNA vaccine. (paper)

  13. Label-free LC-MS analysis of HER2+ breast cancer cell line response to HER2 inhibitor treatment.

    Science.gov (United States)

    Di Luca, Alessio; Henry, Michael; Meleady, Paula; O'Connor, Robert

    2015-08-04

    Human epidermal growth-factor receptor (HER)-2 is overexpressed in 25 % of breast-cancers and is associated with an aggressive form of the disease with significantly shortened disease free and overall survival. In recent years, the use of HER2-targeted therapies, monoclonal-antibodies and small molecule tyrosine-kinase inhibitors has significantly improved the clinical outcome for HER2-positive breast-cancer patients. However, only a fraction of HER2-amplified patients will respond to therapy and the use of these treatments is often limited by tumour drug insensitivity or resistance and drug toxicities. Currently there is no way to identify likely responders or rational combinations with the potential to improve HER2-focussed treatment outcome. In order to further understand the molecular mechanisms of treatment-response with HER2-inhibitors, we used a highly-optimised and reproducible quantitative label-free LC-MS strategy to characterize the proteomes of HER2-overexpressing breast-cancer cell-lines (SKBR3, BT474 and HCC1954) in response to drug-treatment with HER2-inhibitors (lapatinib, neratinib or afatinib). Following 12 ours treatment with different HER2-inhibitors in the BT474 cell-line; compared to the untreated cells, 16 proteins changed significantly in abundance following lapatinib treatment (1 μM), 21 proteins changed significantly following neratinib treatment (150 nM) and 38 proteins changed significantly following afatinib treatment (150 nM). Whereas following 24 hours treatment with neratinib (200 nM) 46 proteins changed significantly in abundance in the HCC1954 cell-line and 23 proteins in the SKBR3 cell-line compared to the untreated cells. Analysing the data we found that, proteins like trifunctional-enzyme subunit-alpha, mitochondrial; heterogeneous nuclear ribonucleoprotein-R and lamina-associated polypeptide 2, isoform alpha were up-regulated whereas heat shock cognate 71 kDa protein was down-regulated in 3 or more comparisons. This proteomic

  14. Dendritic cell targeted chitosan nanoparticles for nasal DNA immunization against SARS CoV nucleocapsid protein.

    Science.gov (United States)

    Raghuwanshi, Dharmendra; Mishra, Vivek; Das, Dipankar; Kaur, Kamaljit; Suresh, Mavanur R

    2012-04-02

    This work investigates the formulation and in vivo efficacy of dendritic cell (DC) targeted plasmid DNA loaded biotinylated chitosan nanoparticles for nasal immunization against nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus (SARS-CoV) as antigen. The induction of antigen-specific mucosal and systemic immune response at the site of virus entry is a major challenge for vaccine design. Here, we designed a strategy for noninvasive receptor mediated gene delivery to nasal resident DCs. The pDNA loaded biotinylated chitosan nanoparticles were prepared using a complex coacervation process and characterized for size, shape, surface charge, plasmid DNA loading and protection against nuclease digestion. The pDNA loaded biotinylated chitosan nanoparticles were targeted with bifunctional fusion protein (bfFp) vector for achieving DC selective targeting. The bfFp is a recombinant fusion protein consisting of truncated core-streptavidin fused with anti-DEC-205 single chain antibody (scFv). The core-streptavidin arm of fusion protein binds with biotinylated nanoparticles, while anti-DEC-205 scFv imparts targeting specificity to DC DEC-205 receptor. We demonstrate that intranasal administration of bfFp targeted formulations along with anti-CD40 DC maturation stimuli enhanced magnitude of mucosal IgA as well as systemic IgG against N protein. The strategy led to the detection of augmented levels of N protein specific systemic IgG and nasal IgA antibodies. However, following intranasal delivery of naked pDNA no mucosal and systemic immune responses were detected. A parallel comparison of targeted formulations using intramuscular and intranasal routes showed that the intramuscular route is superior for induction of systemic IgG responses compared with the intranasal route. Our results suggest that targeted pDNA delivery through a noninvasive intranasal route can be a strategy for designing low-dose vaccines.

  15. Assigning Significance in Label-Free Quantitative Proteomics to Include Single-Peptide-Hit Proteins with Low Replicates

    OpenAIRE

    Li, Qingbo

    2010-01-01

    When sample replicates are limited in a label-free proteomics experiment, selecting differentially regulated proteins with an assignment of statistical significance remains difficult for proteins with a single-peptide hit or a small fold-change. This paper aims to address this issue. An important component of the approach employed here is to utilize the rule of Minimum number of Permuted Significant Pairings (MPSP) to reduce false positives. The MPSP rule generates permuted sample pairings fr...

  16. Computational optimisation of targeted DNA sequencing for cancer detection

    DEFF Research Database (Denmark)

    Martinez, Pierre; McGranahan, Nicholas; Birkbak, Nicolai Juul

    2013-01-01

    Despite recent progress thanks to next-generation sequencing technologies, personalised cancer medicine is still hampered by intra-tumour heterogeneity and drug resistance. As most patients with advanced metastatic disease face poor survival, there is need to improve early diagnosis. Analysing...... detection. Dividing 4,467 samples into one discovery and two independent validation cohorts, we show that up to 76% of 10 cancer types harbour at least one mutation in a panel of only 25 genes, with high sensitivity across most tumour types. Our analyses demonstrate that targeting "hotspot" regions would...

  17. LFQuant: a label-free fast quantitative analysis tool for high-resolution LC-MS/MS proteomics data.

    Science.gov (United States)

    Zhang, Wei; Zhang, Jiyang; Xu, Changming; Li, Ning; Liu, Hui; Ma, Jie; Zhu, Yunping; Xie, Hongwei

    2012-12-01

    Database searching based methods for label-free quantification aim to reconstruct the peptide extracted ion chromatogram based on the identification information, which can limit the search space and thus make the data processing much faster. The random effect of the MS/MS sampling can be remedied by cross-assignment among different runs. Here, we present a new label-free fast quantitative analysis tool, LFQuant, for high-resolution LC-MS/MS proteomics data based on database searching. It is designed to accept raw data in two common formats (mzXML and Thermo RAW), and database search results from mainstream tools (MASCOT, SEQUEST, and X!Tandem), as input data. LFQuant can handle large-scale label-free data with fractionation such as SDS-PAGE and 2D LC. It is easy to use and provides handy user interfaces for data loading, parameter setting, quantitative analysis, and quantitative data visualization. LFQuant was compared with two common quantification software packages, MaxQuant and IDEAL-Q, on the replication data set and the UPS1 standard data set. The results show that LFQuant performs better than them in terms of both precision and accuracy, and consumes significantly less processing time. LFQuant is freely available under the GNU General Public License v3.0 at http://sourceforge.net/projects/lfquant/. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. Live Imaging of Cellular Internalization of Single Colloidal Particle by Combined Label-Free and Fluorescence Total Internal Reflection Microscopy.

    Science.gov (United States)

    Byrne, Gerard D; Vllasaliu, Driton; Falcone, Franco H; Somekh, Michael G; Stolnik, Snjezana

    2015-11-02

    In this work we utilize the combination of label-free total internal reflection microscopy and total internal reflectance fluorescence (TIRM/TIRF) microscopy to achieve a simultaneous, live imaging of single, label-free colloidal particle endocytosis by individual cells. The TIRM arm of the microscope enables label free imaging of the colloid and cell membrane features, while the TIRF arm images the dynamics of fluorescent-labeled clathrin (protein involved in endocytosis via clathrin pathway), expressed in transfected 3T3 fibroblasts cells. Using a model polymeric colloid and cells with a fluorescently tagged clathrin endocytosis pathway, we demonstrate that wide field TIRM/TIRF coimaging enables live visualization of the process of colloidal particle interaction with the labeled cell structure, which is valuable for discerning the membrane events and route of colloid internalization by the cell. We further show that 500 nm in diameter model polystyrene colloid associates with clathrin, prior to and during its cellular internalization. This association is not apparent with larger, 1 μm in diameter colloids, indicating an upper particle size limit for clathrin-mediated endocytosis.

  19. Dynamic nano-imaging of label-free living cells using electron beam excitation-assisted optical microscope

    Science.gov (United States)

    Fukuta, Masahiro; Kanamori, Satoshi; Furukawa, Taichi; Nawa, Yasunori; Inami, Wataru; Lin, Sheng; Kawata, Yoshimasa; Terakawa, Susumu

    2015-01-01

    Optical microscopes are effective tools for cellular function analysis because biological cells can be observed non-destructively and non-invasively in the living state in either water or atmosphere condition. Label-free optical imaging technique such as phase-contrast microscopy has been analysed many cellular functions, and it is essential technology for bioscience field. However, the diffraction limit of light makes it is difficult to image nano-structures in a label-free living cell, for example the endoplasmic reticulum, the Golgi body and the localization of proteins. Here we demonstrate the dynamic imaging of a label-free cell with high spatial resolution by using an electron beam excitation-assisted optical (EXA) microscope. We observed the dynamic movement of the nucleus and nano-scale granules in living cells with better than 100 nm spatial resolution and a signal-to-noise ratio (SNR) around 10. Our results contribute to the development of cellular function analysis and open up new bioscience applications. PMID:26525841

  20. Dynamic nano-imaging of label-free living cells using electron beam excitation-assisted optical microscope.

    Science.gov (United States)

    Fukuta, Masahiro; Kanamori, Satoshi; Furukawa, Taichi; Nawa, Yasunori; Inami, Wataru; Lin, Sheng; Kawata, Yoshimasa; Terakawa, Susumu

    2015-11-03

    Optical microscopes are effective tools for cellular function analysis because biological cells can be observed non-destructively and non-invasively in the living state in either water or atmosphere condition. Label-free optical imaging technique such as phase-contrast microscopy has been analysed many cellular functions, and it is essential technology for bioscience field. However, the diffraction limit of light makes it is difficult to image nano-structures in a label-free living cell, for example the endoplasmic reticulum, the Golgi body and the localization of proteins. Here we demonstrate the dynamic imaging of a label-free cell with high spatial resolution by using an electron beam excitation-assisted optical (EXA) microscope. We observed the dynamic movement of the nucleus and nano-scale granules in living cells with better than 100 nm spatial resolution and a signal-to-noise ratio (SNR) around 10. Our results contribute to the development of cellular function analysis and open up new bioscience applications.

  1. Whole-organ atlas imaged by label-free high-resolution photoacoustic microscopy assisted by a microtome

    Science.gov (United States)

    Wong, Terence T. W.; Zhang, Ruiying; Hsu, Hsun-Chia; Maslov, Konstantin I.; Shi, Junhui; Chen, Ruimin; Shung, K. Kirk; Zhou, Qifa; Wang, Lihong V.

    2018-02-01

    In biomedical imaging, all optical techniques face a fundamental trade-off between spatial resolution and tissue penetration. Therefore, obtaining an organelle-level resolution image of a whole organ has remained a challenging and yet appealing scientific pursuit. Over the past decade, optical microscopy assisted by mechanical sectioning or chemical clearing of tissue has been demonstrated as a powerful technique to overcome this dilemma, one of particular use in imaging the neural network. However, this type of techniques needs lengthy special preparation of the tissue specimen, which hinders broad application in life sciences. Here, we propose a new label-free three-dimensional imaging technique, named microtomy-assisted photoacoustic microscopy (mPAM), for potentially imaging all biomolecules with 100% endogenous natural staining in whole organs with high fidelity. We demonstrate the first label-free mPAM, using UV light for label-free histology-like imaging, in whole organs (e.g., mouse brains), most of them formalin-fixed and paraffin- or agarose-embedded for minimal morphological deformation. Furthermore, mPAM with dual wavelength illuminations is also employed to image a mouse brain slice, demonstrating the potential for imaging of multiple biomolecules without staining. With visible light illumination, mPAM also shows its deep tissue imaging capability, which enables less slicing and hence reduces sectioning artifacts. mPAM could potentially provide a new insight for understanding complex biological organs.

  2. Long-circulating DNA lipid nanocapsules as new vector for passive tumor targeting.

    OpenAIRE

    Morille , Marie; Montier , Tristan; Legras , Pierre; Carmoy , Nathalie; Brodin , Priscille; Pitard , Bruno; Benoît , Jean-Pierre; Passirani , Catherine

    2010-01-01

    International audience; Systemic gene delivery systems are needed for therapeutic application to organs that are inaccessible by percutaneous injection. Currently, the main objective is the development of a stable and non-toxic vector that can encapsulate and deliver foreign genetic material to target cells. To this end, DNA, complexed with cationic lipids i.e. DOTAP/DOPE, was encapsulated into lipid nanocapsules (LNCs) leading to the formation of stable nanocarriers (DNA LNCs) with a size in...

  3. Potential advantages of DNA methyltransferase 1 (DNMT1)-targeted inhibition for cancer therapy.

    Science.gov (United States)

    Jung, Yeonjoo; Park, Jinah; Kim, Tai Young; Park, Jung-Hyun; Jong, Hyun-Soon; Im, Seock-Ah; Robertson, Keith D; Bang, Yung-Jue; Kim, Tae-You

    2007-10-01

    The deoxyribonucleic acid (DNA) methyltransferase (DNMT) inhibitor 5-aza-2'-deoxycytidine (5-aza-dC) has been used as a drug in a part of cancer therapy. However, because of its incorporation into DNA during DNA synthesis, 5-aza-dC can cause DNA damage, mutagenesis, and cytotoxicity. In view of the adverse effects of 5-aza-dC, DNMT-targeted inhibition may be a more effective approach than treatment with 5-aza-dC. To address the possibility of DNMT-targeted cancer therapy, we compared the effects of treatment with small interfering ribonucleic acids (siRNAs) specific for DNMT1 or DNMT3b and treatment with 5-aza-dC on transcription, cell growth, and DNA damage in gastric cancer cells. We found that DNMT1-targeted inhibition induced the re-expression and reversed DNA methylation of five (CDKN2A, RASSF1A, HTLF, RUNX3, and AKAP12B) out of seven genes examined, and 5-aza-dC reactivated and demethylated all seven genes. In contrast, DNMT3b siRNAs did not show any effect. Furthermore, the double knockdown of DNMT1 and DNMT3b did not show a synergistic effect on gene re-expression and demethylation. In addition, DNMT1 siRNAs showed an inhibitory effect of cell proliferation in the cancer cells and the induction of cell death without evidence of DNA damage, whereas treatment with 5-aza-dC caused DNA damage as demonstrated by the comet assay. These results provide a rationale for the development of a DNMT1-targeted strategy as an effective epigenetic cancer therapy.

  4. Fluorometric detection of adenine in target DNA by exciplex formation with fluorescent 8-arylethynylated deoxyguanosine.

    Science.gov (United States)

    Saito, Yoshio; Kugenuma, Kenji; Tanaka, Makiko; Suzuki, Azusa; Saito, Isao

    2012-06-01

    We demonstrated an intriguing method to discriminate adenine by incident appearance of an intense new emission via exciplex formation in hybridization of target DNA with newly designed fluorescent 8-arylethynylated deoxyguanosine derivatives. We described the synthesis of such highly electron donating fluorescent guanosine derivatives and their incorporation into DNA oligomers which may be used for the structural study and the fluorometric analysis of nucleic acids. Copyright © 2012 Elsevier Ltd. All rights reserved.

  5. DNA-Dependent Protein Kinase As Molecular Target for Radiosensitization of Neuroblastoma Cells.

    Directory of Open Access Journals (Sweden)

    M Emmy M Dolman

    Full Text Available Tumor cells might resist therapy with ionizing radiation (IR by non-homologous end-joining (NHEJ of IR-induced double-strand breaks. One of the key players in NHEJ is DNA-dependent protein kinase (DNA-PK. The catalytic subunit of DNA-PK, i.e. DNA-PKcs, can be inhibited with the small-molecule inhibitor NU7026. In the current study, the in vitro potential of NU7026 to radiosensitize neuroblastoma cells was investigated. DNA-PKcs is encoded by the PRKDC (protein kinase, DNA-activated, catalytic polypeptide gene. We showed that PRKDC levels were enhanced in neuroblastoma patients and correlated with a more advanced tumor stage and poor prognosis, making DNA-PKcs an interesting target for radiosensitization of neuroblastoma tumors. Optimal dose finding for combination treatment with NU7026 and IR was performed using NGP cells. One hour pre-treatment with 10 μM NU7026 synergistically sensitized NGP cells to 0.63 Gy IR. Radiosensitizing effects of NU7026 increased in time, with maximum effects observed from 96 h after IR-exposure on. Combined treatment of NGP cells with 10 μM NU7026 and 0.63 Gy IR resulted in apoptosis, while no apoptotic response was observed for either of the therapies alone. Inhibition of IR-induced DNA-PK activation by NU7026 confirmed the capability of NGP cells to, at least partially, resist IR by NHEJ. NU7026 also synergistically radiosensitized other neuroblastoma cell lines, while no synergistic effect was observed for low DNA-PKcs-expressing non-cancerous fibroblasts. Results obtained for NU7026 were confirmed by PRKDC knockdown in NGP cells. Taken together, the current study shows that DNA-PKcs is a promising target for neuroblastoma radiosensitization.

  6. DNA-Dependent Protein Kinase As Molecular Target for Radiosensitization of Neuroblastoma Cells.

    Science.gov (United States)

    Dolman, M Emmy M; van der Ploeg, Ida; Koster, Jan; Bate-Eya, Laurel Tabe; Versteeg, Rogier; Caron, Huib N; Molenaar, Jan J

    2015-01-01

    Tumor cells might resist therapy with ionizing radiation (IR) by non-homologous end-joining (NHEJ) of IR-induced double-strand breaks. One of the key players in NHEJ is DNA-dependent protein kinase (DNA-PK). The catalytic subunit of DNA-PK, i.e. DNA-PKcs, can be inhibited with the small-molecule inhibitor NU7026. In the current study, the in vitro potential of NU7026 to radiosensitize neuroblastoma cells was investigated. DNA-PKcs is encoded by the PRKDC (protein kinase, DNA-activated, catalytic polypeptide) gene. We showed that PRKDC levels were enhanced in neuroblastoma patients and correlated with a more advanced tumor stage and poor prognosis, making DNA-PKcs an interesting target for radiosensitization of neuroblastoma tumors. Optimal dose finding for combination treatment with NU7026 and IR was performed using NGP cells. One hour pre-treatment with 10 μM NU7026 synergistically sensitized NGP cells to 0.63 Gy IR. Radiosensitizing effects of NU7026 increased in time, with maximum effects observed from 96 h after IR-exposure on. Combined treatment of NGP cells with 10 μM NU7026 and 0.63 Gy IR resulted in apoptosis, while no apoptotic response was observed for either of the therapies alone. Inhibition of IR-induced DNA-PK activation by NU7026 confirmed the capability of NGP cells to, at least partially, resist IR by NHEJ. NU7026 also synergistically radiosensitized other neuroblastoma cell lines, while no synergistic effect was observed for low DNA-PKcs-expressing non-cancerous fibroblasts. Results obtained for NU7026 were confirmed by PRKDC knockdown in NGP cells. Taken together, the current study shows that DNA-PKcs is a promising target for neuroblastoma radiosensitization.

  7. Fluorescence turn-on detection of target sequence DNA based on silicon nanodot-mediated quenching.

    Science.gov (United States)

    Zhang, Yanan; Ning, Xinping; Mao, Guobin; Ji, Xinghu; He, Zhike

    2018-05-01

    We have developed a new enzyme-free method for target sequence DNA detection based on the dynamic quenching of fluorescent silicon nanodots (SiNDs) toward Cy5-tagged DNA probe. Fascinatingly, the water-soluble SiNDs can quench the fluorescence of cyanine (Cy5) in Cy5-tagged DNA probe in homogeneous solution, and the fluorescence of Cy5-tagged DNA probe can be restored in the presence of target sequence DNA (the synthetic target miRNA-27a). Based on this phenomenon, a SiND-featured fluorescent sensor has been constructed for "turn-on" detection of the synthetic target miRNA-27a for the first time. This newly developed approach possesses the merits of low cost, simple design, and convenient operation since no enzymatic reaction, toxic reagents, or separation procedures are involved. The established method achieves a detection limit of 0.16 nM, and the relative standard deviation of this method is 9% (1 nM, n = 5). The linear range is 0.5-20 nM, and the recoveries in spiked human fluids are in the range of 90-122%. This protocol provides a new tactic in the development of the nonenzymic miRNA biosensors and opens a promising avenue for early diagnosis of miRNA-associated disease. Graphical abstract The SiND-based fluorescent sensor for detection of S-miR-27a.

  8. Radiosensitizing and cytotoxic properties of DNA targeted phenanthridine-linked nitroheterocycles of varying electron affinities

    International Nuclear Information System (INIS)

    Cowan, D.S.M.; Rauth, A.M.; Toronto Univ., ON; Matejovic, J.F.; McClelland, R.A.; Wardman, P.

    1994-01-01

    2-Nitroimidazoles targeted to DNA via intercalation have previously been shown to be as much as 10-100 times more efficient on a molar basis than the untargeted nitroimidazole, misonidazole, in vitro as hypoxic cell selective radiosensitizers and cytotoxins based on extracellular concentrations. In this work the effect of varying the nitroaromatic group has been examined through the preparation of a DNA-targeted 4-nitroimidazole (4-MeNLP-3), a 5-nitroimidazole (5-NLP-3) and a 5-nitrofuran (FEP-2) linked to phenanthridinium ions. With the previously synthesized 2-nitroimidazoles, this provides a series of DNA targeted compounds of varying electron affinity as well as structure at the nitroaromatic position. The present series of compounds was tested for partition coefficient, DNA binding ability, reduction potentials and in vitro radiosensitizing and cytotoxic abilities. The results obtained indicate that targeting such compounds to DNA diminishes the dependency of radiosensitizing and cytotoxic properties on reduction potential and may allow significant uncoupling of toxicity from radiosensitizing ability. (author)

  9. A label-free quantitative shotgun proteomics analysis of rice grain development

    Directory of Open Access Journals (Sweden)

    Koh Hee-Jong

    2011-09-01

    Full Text Available Abstract Background Although a great deal of rice proteomic research has been conducted, there are relatively few studies specifically addressing the rice grain proteome. The existing rice grain proteomic researches have focused on the identification of differentially expressed proteins or monitoring protein expression patterns during grain filling stages. Results Proteins were extracted from rice grains 10, 20, and 30 days after flowering, as well as from fully mature grains. By merging all of the identified proteins in this study, we identified 4,172 non-redundant proteins with a wide range of molecular weights (from 5.2 kDa to 611 kDa and pI values (from pH 2.9 to pH 12.6. A Genome Ontology category enrichment analysis for the 4,172 proteins revealed that 52 categories were enriched, including the carbohydrate metabolic process, transport, localization, lipid metabolic process, and secondary metabolic process. The relative abundances of the 1,784 reproducibly identified proteins were compared to detect 484 differentially expressed proteins during rice grain development. Clustering analysis and Genome Ontology category enrichment analysis revealed that proteins involved in the metabolic process were enriched through all stages of development, suggesting that proteome changes occurred even in the desiccation phase. Interestingly, enrichments of proteins involved in protein folding were detected in the desiccation phase and in fully mature grain. Conclusion This is the first report conducting comprehensive identification of rice grain proteins. With a label free shotgun proteomic approach, we identified large number of rice grain proteins and compared the expression patterns of reproducibly identified proteins during rice grain development. Clustering analysis, Genome Ontology category enrichment analysis, and the analysis of composite expression profiles revealed dynamic changes of metabolisms during rice grain development. Interestingly, we

  10. Label-Free Analysis of Cellular Lipid Droplet Formation by Non-Linear Microscopy

    Science.gov (United States)

    Schie, Iwan W.

    Cellular lipid droplets (LD) are cellular organelles that can be found in every cell type. Recent research indicates that cellular LD are involved in a large number of cellular metabolic functions, such as lipid metabolism, protection from lipotoxicity, protein storage and degradation, and many more. LD formation is frequently associated with adverse health effects, i.e. alcoholic and non-alcoholic fatty liver disease, diabetes type-2, as well as many cardiovascular disorders. Despite their wide presence, LDs are the least studied and most poorly understood cellular organelles. Typically, LDs are investigated using fluorescence-based techniques that require staining with exogenous fluorophores. Other techniques, e.g. biochemical assays, require the destruction of cells that prohibit the analysis of living cells. Therefore, in my thesis research I developed a novel compound fast-scanning nonlinear optical microscope equipped with the ability to also acquire Raman spectra at specific image locations. This system allows us to image label-free cellular LD formation in living cells and analyze the composition of single cellular LDs. Images can be acquired at near video-rate (˜16 frames/s). Furthermore, the system has the ability to acquire very large images of tissue of up to 7.5x15 cm2 total area by stitching together scans with dimensions of 1x1 mm2 in less than 1 minute. The system also enables the user to acquire Raman spectra from points of interest in the multiphoton images and provides chemically-specific data from sample volumes as small as 1 femtoliter. In my thesis I used this setup to determine the effects of VLDL lipolysis products on primary rat hepatocytes. By analyzing the Raman spectra and comparing the peak ratios for saturated and unsaturated fatty acid it was determined that the small cellular LD are highly saturated, while large cellular LDs contain mostly unsaturated lipids. Furthermore, I established a method to determine the specific contribution

  11. Germline Variants in Targeted Tumor Sequencing Using Matched Normal DNA.

    Science.gov (United States)

    Schrader, Kasmintan A; Cheng, Donavan T; Joseph, Vijai; Prasad, Meera; Walsh, Michael; Zehir, Ahmet; Ni, Ai; Thomas, Tinu; Benayed, Ryma; Ashraf, Asad; Lincoln, Annie; Arcila, Maria; Stadler, Zsofia; Solit, David; Hyman, David M; Hyman, David; Zhang, Liying; Klimstra, David; Ladanyi, Marc; Offit, Kenneth; Berger, Michael; Robson, Mark

    2016-01-01

    Tumor genetic sequencing identifies potentially targetable genetic alterations with therapeutic implications. Analysis has concentrated on detecting tumor-specific variants, but recognition of germline variants may prove valuable as well. To estimate the burden of germline variants identified through routine clinical tumor sequencing. Patients with advanced cancer diagnoses eligible for studies of targeted agents at Memorial Sloan Kettering Cancer Center are offered tumor-normal sequencing with MSK-IMPACT, a 341-gene panel. We surveyed the germline variants seen in 187 overlapping genes with Mendelian disease associations in 1566 patients who had undergone tumor profiling between March and October 2014. The number of presumed pathogenic germline variants (PPGVs) and variants of uncertain significance per person in 187 genes associated with single-gene disorders and the proportions of individuals with PPGVs in clinically relevant gene subsets, in genes consistent with known tumor phenotypes, and in genes with evidence of second somatic hits in their tumors. The mean age of the 1566 patients was 58 years, and 54% were women. Presumed pathogenic germline variants in known Mendelian disease-associated genes were identified in 246 of 1566 patients (15.7%; 95% CI, 14.0%-17.6%), including 198 individuals with mutations in genes associated with cancer susceptibility. Germline findings in cancer susceptibility genes were concordant with the individual's cancer type in only 81 of 198 cases (40.9%; 95% CI, 34.3%-47.9%). In individuals with PPGVs retained in the tumor, somatic alteration of the other allele was seen in 39 of 182 cases (21.4%; 95% CI, 16.1%-28.0%), of which 13 cases did not show a known correlation of the germline mutation and a known syndrome. Mutations in non-cancer-related Mendelian disease genes were seen in 55 of 1566 cases (3.5%; 95% CI, 27.1%-45.4%). Almost every individual had more than 1 variant of uncertain significance (1565 of 1566 patients; 99

  12. Evaluating Digital PCR for the Quantification of Human Genomic DNA: Accessible Amplifiable Targets.

    Science.gov (United States)

    Kline, Margaret C; Romsos, Erica L; Duewer, David L

    2016-02-16

    Polymerase chain reaction (PCR) multiplexed assays perform best when the input quantity of template DNA is controlled to within about a factor of √2. To help ensure that PCR assays yield consistent results over time and place, results from methods used to determine DNA quantity need to be metrologically traceable to a common reference. Many DNA quantitation systems can be accurately calibrated with solutions of DNA in aqueous buffer. Since they do not require external calibration, end-point limiting dilution technologies, collectively termed "digital PCR (dPCR)", have been proposed as suitable for value assigning such DNA calibrants. The performance characteristics of several commercially available dPCR systems have recently been documented using plasmid, viral, or fragmented genomic DNA; dPCR performance with more complex materials, such as human genomic DNA, has been less studied. With the goal of providing a human genomic reference material traceably certified for mass concentration, we are investigating the measurement characteristics of several dPCR systems. We here report results of measurements from multiple PCR assays, on four human genomic DNAs treated with four endonuclease restriction enzymes using both chamber and droplet dPCR platforms. We conclude that dPCR does not estimate the absolute number of PCR targets in a given volume but rather the number of accessible and amplifiable targets. While enzymatic restriction of human genomic DNA increases accessibility for some assays, in well-optimized PCR assays it can reduce the number of amplifiable targets and increase assay variability relative to uncut sample.

  13. Functional DNA-containing nanomaterials: cellular applications in biosensing, imaging, and targeted therapy.

    Science.gov (United States)

    Liang, Hao; Zhang, Xiao-Bing; Lv, Yifan; Gong, Liang; Wang, Ruowen; Zhu, Xiaoyan; Yang, Ronghua; Tan, Weihong

    2014-06-17

    CONSPECTUS: DNA performs a vital function as a carrier of genetic code, but in the field of nanotechnology, DNA molecules can catalyze chemical reactions in the cell, that is, DNAzymes, or bind with target-specific ligands, that is, aptamers. These functional DNAs with different modifications have been developed for sensing, imaging, and therapeutic systems. Thus, functional DNAs hold great promise for future applications in nanotechnology and bioanalysis. However, these functional DNAs face challenges, especially in the field of biomedicine. For example, functional DNAs typically require the use of cationic transfection reagents to realize cellular uptake. Such reagents enter the cells, increasing the difficulty of performing bioassays in vivo and potentially damaging the cell's nucleus. To address this obstacle, nanomaterials, such as metallic, carbon, silica, or magnetic materials, have been utilized as DNA carriers or assistants. In this Account, we describe selected examples of functional DNA-containing nanomaterials and their applications from our recent research and those of others. As models, we have chosen to highlight DNA/nanomaterial complexes consisting of gold nanoparticles, graphene oxides, and aptamer-micelles, and we illustrate the potential of such complexes in biosensing, imaging, and medical diagnostics. Under proper conditions, multiple ligand-receptor interactions, decreased steric hindrance, and increased surface roughness can be achieved from a high density of DNA that is bound to the surface of nanomaterials, resulting in a higher affinity for complementary DNA and other targets. In addition, this high density of DNA causes a high local salt concentration and negative charge density, which can prevent DNA degradation. For example, DNAzymes assembled on gold nanoparticles can effectively catalyze chemical reactions even in living cells. And it has been confirmed that DNA-nanomaterial complexes can enter cells more easily than free single

  14. Multiplex target enrichment using DNA indexing for ultra-high throughput SNP detection.

    LENUS (Irish Health Repository)

    Kenny, Elaine M

    2011-02-01

    Screening large numbers of target regions in multiple DNA samples for sequence variation is an important application of next-generation sequencing but an efficient method to enrich the samples in parallel has yet to be reported. We describe an advanced method that combines DNA samples using indexes or barcodes prior to target enrichment to facilitate this type of experiment. Sequencing libraries for multiple individual DNA samples, each incorporating a unique 6-bp index, are combined in equal quantities, enriched using a single in-solution target enrichment assay and sequenced in a single reaction. Sequence reads are parsed based on the index, allowing sequence analysis of individual samples. We show that the use of indexed samples does not impact on the efficiency of the enrichment reaction. For three- and nine-indexed HapMap DNA samples, the method was found to be highly accurate for SNP identification. Even with sequence coverage as low as 8x, 99% of sequence SNP calls were concordant with known genotypes. Within a single experiment, this method can sequence the exonic regions of hundreds of genes in tens of samples for sequence and structural variation using as little as 1 μg of input DNA per sample.

  15. Identification and Characterization of a Small Inhibitory Peptide That Can Target DNA-PKcs Autophosphorylation and Increase Tumor Radiosensitivity

    Energy Technology Data Exchange (ETDEWEB)

    Sun Xiaonan [Department of Radiation Oncology, Sir Run Run Shaw Hospital, Sir Run Run Shaw Institute of Clinical Medicine of Zhejiang University, Hangzhou (China); Yang Chunying [Department of Radiation Oncology, Methodist Hospital Research Institute, Weill Cornell Medical College, Houston, TX (United States); Liu Hai; Wang Qi [Department of Radiation Oncology, Sir Run Run Shaw Hospital, Sir Run Run Shaw Institute of Clinical Medicine of Zhejiang University, Hangzhou (China); Wu Shixiu [Department of Radiation Oncology, The First Affiliated Hospital of Wenzhou Medical College, Wenzhou (China); Li Xia; Xie Tian [Research Center of Biomedicine and Health, Hangzhou Normal University, Hangzhou (China); Brinkman, Kathryn L.; Teh, Bin S.; Butler, E. Brian [Department of Radiation Oncology, Methodist Hospital Research Institute, Weill Cornell Medical College, Houston, TX (United States); Xu Bo, E-mail: bxu@tmhs.org [Department of Radiation Oncology, Methodist Hospital Research Institute, Weill Cornell Medical College, Houston, TX (United States); Zheng, Shu, E-mail: zhengshu@zju.edu.cn [Cancer Institute, The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou (China)

    2012-12-01

    Purpose: The DNA protein kinase catalytic subunit (DNA-PKcs) is one of the critical elements involved in the DNA damage repair process. Inhibition of DNA-PKcs results in hypersensitivity to ionizing radiation (IR); therefore, this approach has been explored to develop molecular targeted radiosensitizers. Here, we aimed to develop small inhibitory peptides that could specifically target DNA-PKcs autophosphorylation, a critical step for the enzymatic activation of the kinase in response to IR. Methods and Materials: We generated several small fusion peptides consisting of 2 functional domains, 1 an internalization domain and the other a DNA-PKcs autophosphorylation inhibitory domain. We characterized the internalization, toxicity, and radiosensitization activities of the fusion peptides. Furthermore, we studied the mechanisms of the inhibitory peptides on DNA-PKcs autophosphorylation and DNA repair. Results: We found that among several peptides, the biotin-labeled peptide 3 (BTW3) peptide, which targets DNA-PKcs threonine 2647 autophosphorylation, can abrogate IR-induced DNA-PKcs activation and cause prolonged {gamma}-H2AX focus formation. We demonstrated that BTW3 exposure led to hypersensitivity to IR in DNA-PKcs-proficient cells but not in DNA-PKcs-deficient cells. Conclusions: The small inhibitory peptide BTW3 can specifically target DNA-PKcs autophosphorylation and enhance radiosensitivity; therefore, it can be further developed as a novel class of radiosensitizer.

  16. Identification and Characterization of a Small Inhibitory Peptide That Can Target DNA-PKcs Autophosphorylation and Increase Tumor Radiosensitivity

    International Nuclear Information System (INIS)

    Sun Xiaonan; Yang Chunying; Liu Hai; Wang Qi; Wu Shixiu; Li Xia; Xie Tian; Brinkman, Kathryn L.; Teh, Bin S.; Butler, E. Brian; Xu Bo; Zheng, Shu

    2012-01-01

    Purpose: The DNA protein kinase catalytic subunit (DNA-PKcs) is one of the critical elements involved in the DNA damage repair process. Inhibition of DNA-PKcs results in hypersensitivity to ionizing radiation (IR); therefore, this approach has been explored to develop molecular targeted radiosensitizers. Here, we aimed to develop small inhibitory peptides that could specifically target DNA-PKcs autophosphorylation, a critical step for the enzymatic activation of the kinase in response to IR. Methods and Materials: We generated several small fusion peptides consisting of 2 functional domains, 1 an internalization domain and the other a DNA-PKcs autophosphorylation inhibitory domain. We characterized the internalization, toxicity, and radiosensitization activities of the fusion peptides. Furthermore, we studied the mechanisms of the inhibitory peptides on DNA-PKcs autophosphorylation and DNA repair. Results: We found that among several peptides, the biotin-labeled peptide 3 (BTW3) peptide, which targets DNA-PKcs threonine 2647 autophosphorylation, can abrogate IR-induced DNA-PKcs activation and cause prolonged γ-H2AX focus formation. We demonstrated that BTW3 exposure led to hypersensitivity to IR in DNA-PKcs-proficient cells but not in DNA-PKcs-deficient cells. Conclusions: The small inhibitory peptide BTW3 can specifically target DNA-PKcs autophosphorylation and enhance radiosensitivity; therefore, it can be further developed as a novel class of radiosensitizer.

  17. On Orbit Immuno-Based, Label-Free, White Blood Cell Counting System with MicroElectroMechanical Sensor (MEMS) Technology (OILWBCS-MEMS), Phase II

    Data.gov (United States)

    National Aeronautics and Space Administration — Aurora Flight Sciences Corporation and partner, Draper Laboratory, propose to develop an on-orbit immuno-based label-free white blood cell counting system using MEMS...

  18. On Orbit Immuno-Based, Label-Free, White Blood Cell Counting System with MicroElectroMechanical Sensor (MEMS) Technology (OILWBCS-MEMS), Phase I

    Data.gov (United States)

    National Aeronautics and Space Administration — Aurora Flight Sciences Corporation and our partner, Draper Laboratory, propose to develop an on orbit immuno-based, label-free, white blood cell counting system for...

  19. DNA demethylases target promoter transposable elements to positively regulate stress responsive genes in Arabidopsis.

    Science.gov (United States)

    Le, Tuan-Ngoc; Schumann, Ulrike; Smith, Neil A; Tiwari, Sameer; Au, Phil Chi Khang; Zhu, Qian-Hao; Taylor, Jennifer M; Kazan, Kemal; Llewellyn, Danny J; Zhang, Ren; Dennis, Elizabeth S; Wang, Ming-Bo

    2014-09-17

    DNA demethylases regulate DNA methylation levels in eukaryotes. Arabidopsis encodes four DNA demethylases, DEMETER (DME), REPRESSOR OF SILENCING 1 (ROS1), DEMETER-LIKE 2 (DML2), and DML3. While DME is involved in maternal specific gene expression during seed development, the biological function of the remaining DNA demethylases remains unclear. We show that ROS1, DML2, and DML3 play a role in fungal disease resistance in Arabidopsis. A triple DNA demethylase mutant, rdd (ros1 dml2 dml3), shows increased susceptibility to the fungal pathogen Fusarium oxysporum. We identify 348 genes differentially expressed in rdd relative to wild type, and a significant proportion of these genes are downregulated in rdd and have functions in stress response, suggesting that DNA demethylases maintain or positively regulate the expression of stress response genes required for F. oxysporum resistance. The rdd-downregulated stress response genes are enriched for short transposable element sequences in their promoters. Many of these transposable elements and their surrounding sequences show localized DNA methylation changes in rdd, and a general reduction in CHH methylation, suggesting that RNA-directed DNA methylation (RdDM), responsible for CHH methylation, may participate in DNA demethylase-mediated regulation of stress response genes. Many of the rdd-downregulated stress response genes are downregulated in the RdDM mutants nrpd1 and nrpe1, and the RdDM mutants nrpe1 and ago4 show enhanced susceptibility to F. oxysporum infection. Our results suggest that a primary function of DNA demethylases in plants is to regulate the expression of stress response genes by targeting promoter transposable element sequences.

  20. The heat-transfer method: a versatile low-cost, label-free, fast, and user-friendly readout platform for biosensor applications.

    Science.gov (United States)

    van Grinsven, Bart; Eersels, Kasper; Peeters, Marloes; Losada-Pérez, Patricia; Vandenryt, Thijs; Cleij, Thomas J; Wagner, Patrick

    2014-08-27

    In recent years, biosensors have become increasingly important in various scientific domains including medicine, biology, and pharmacology, resulting in an increased demand for fast and effective readout techniques. In this Spotlight on Applications, we report on the recently developed heat-transfer method (HTM) and illustrate the use of the technique by zooming in on four established bio(mimetic) sensor applications: (i) mutation analysis in DNA sequences, (ii) cancer cell identification through surface-imprinted polymers, (iii) detection of neurotransmitters with molecularly imprinted polymers, and (iv) phase-transition analysis in lipid vesicle layers. The methodology is based on changes in heat-transfer resistance at a functionalized solid-liquid interface. To this extent, the device applies a temperature gradient over this interface and monitors the temperature underneath and above the functionalized chip in time. The heat-transfer resistance can be obtained by dividing this temperature gradient by the power needed to achieve a programmed temperature. The low-cost, fast, label-free and user-friendly nature of the technology in combination with a high degree of specificity, selectivity, and sensitivity makes HTM a promising sensor technology.

  1. Label-free Proteomic Reveals that Cowpea Severe Mosaic Virus Transiently Suppresses the Host Leaf Protein Accumulation During the Compatible Interaction with Cowpea (Vigna unguiculata [L.] Walp.).

    Science.gov (United States)

    Paiva, Ana L S; Oliveira, Jose T A; de Souza, Gustavo A; Vasconcelos, Ilka M

    2016-12-02

    Viruses are important plant pathogens that threaten diverse crops worldwide. Diseases caused by Cowpea severe mosaic virus (CPSMV) have drawn attention because of the serious damages they cause to economically important crops including cowpea. This work was undertaken to quantify and identify the responsive proteins of a susceptible cowpea genotype infected with CPSMV, in comparison with mock-inoculated controls, using label-free quantitative proteomics and databanks, aiming at providing insights on the molecular basis of this compatible interaction. Cowpea leaves were mock- or CPSMV-inoculated and 2 and 6 days later proteins were extracted and analyzed. More than 3000 proteins were identified (data available via ProteomeXchange, identifier PXD005025) and 75 and 55 of them differentially accumulated in response to CPSMV, at 2 and 6 DAI, respectively. At 2 DAI, 76% of the proteins decreased in amount and 24% increased. However, at 6 DAI, 100% of the identified proteins increased. Thus, CPSMV transiently suppresses the synthesis of proteins involved particularly in the redox homeostasis, protein synthesis, defense, stress, RNA/DNA metabolism, signaling, and other functions, allowing viral invasion and spread in cowpea tissues.

  2. Infrared microspectroscopy of live cells in microfluidic devices (MD-IRMS): toward a powerful label-free cell-based assay.

    Science.gov (United States)

    Vaccari, L; Birarda, G; Businaro, L; Pacor, S; Grenci, G

    2012-06-05

    Until nowadays most infrared microspectroscopy (IRMS) experiments on biological specimens (i.e., tissues or cells) have been routinely carried out on fixed or dried samples in order to circumvent water absorption problems. In this paper, we demonstrate the possibility to widen the range of in-vitro IRMS experiments to vibrational analysis of live cellular samples, thanks to the development of novel biocompatible IR-visible transparent microfluidic devices (MD). In order to highlight the biological relevance of IRMS in MD (MD-IRMS), we performed a systematic exploration of the biochemical alterations induced by different fixation protocols, ethanol 70% and formaldehyde solution 4%, as well as air-drying on U937 leukemic monocytes by comparing their IR vibrational features with the live U937 counterpart. Both fixation and air-drying procedures affected lipid composition and order as well as protein structure at a different extent while they both induced structural alterations in nucleic acids. Therefore, only IRMS of live cells can provide reliable information on both DNA and RNA structure and on their cellular dynamic. In summary, we show that MD-IRMS of live cells is feasible, reliable, and biologically relevant to be recognized as a label-free cell-based assay.

  3. DNA-templated antibody conjugation for targeted drug delivery to cancer cells

    DEFF Research Database (Denmark)

    Liu, Tianqiang

    2016-01-01

    -templated organic synthesis due to the wide existence of the 3-histidine cluster in most wild-type proteins. In this thesis, three projects that relate to targeted drug delivery to cancer cells based on the DTPC method is described. The first project was a delivery system which uses transferrin as the targeting....... The study shows that DNA is a highly useful tool for the assembly of proteins with potential therapeutic applications. The DNA-templated protein conjugation shows a promising application in constructing antibody-toxin conjugates or antibody-drug conjugates. In addition, DNA strands used for antibody...... either antibody engineering or special expression systems and are both time and labor consuming. To avoid the drawbacks caused by conventional chemical modification and recombinant methodologies, an ideal site specific conjugation technique would use natural amino acid residues to the protein by a new...

  4. A multi-domain protein for beta1 integrin-targeted DNA delivery.

    NARCIS (Netherlands)

    E. Fortunati (Elisabetta); E.M.E. Ehlert (Ehrich); N.D. van Loo; C. Wyman (Claire); J.A. Eble; F.G. Grosveld (Frank); B.J. Scholte (Bob)

    2000-01-01

    textabstractThe development of effective receptor-targeted nonviral vectors for use in vivo is complicated by a number of technical problems. One of these is the low efficiency of the conjugation procedures used to couple protein ligands to the DNA condensing carrier molecules. We have made and

  5. Is DNA Alive? a Study of Conceptual Change through Targeted Instruction

    Science.gov (United States)

    Witzig, Stephen B.; Freyermuth, Sharyn K.; Siegel, Marcelle A.; Izci, Kemal; Pires, J. Chris

    2013-01-01

    We are involved in a project to incorporate innovative assessments within a reform-based large-lecture biochemistry course for nonmajors. We not only assessed misconceptions but purposefully changed instruction throughout the semester to confront student ideas. Our research questions targeted student conceptions of deoxyribonucleic acid (DNA)…

  6. Detection of genetically modified organisms (GMOs) using isothermal amplification of target DNA sequences.

    Science.gov (United States)

    Lee, David; La Mura, Maurizio; Allnutt, Theo R; Powell, Wayne

    2009-02-02

    The most common method of GMO detection is based upon the amplification of GMO-specific DNA amplicons using the polymerase chain reaction (PCR). Here we have applied the loop-mediated isothermal amplification (LAMP) method to amplify GMO-related DNA sequences, 'internal' commonly-used motifs for controlling transgene expression and event-specific (plant-transgene) junctions. We have tested the specificity and sensitivity of the technique for use in GMO studies. Results show that detection of 0.01% GMO in equivalent background DNA was possible and dilutions of template suggest that detection from single copies of the template may be possible using LAMP. This work shows that GMO detection can be carried out using LAMP for routine screening as well as for specific events detection. Moreover, the sensitivity and ability to amplify targets, even with a high background of DNA, here demonstrated, highlights the advantages of this isothermal amplification when applied for GMO detection.

  7. High Throughput, Label-free Screening Small Molecule Compound Libraries for Protein-Ligands using Combination of Small Molecule Microarrays and a Special Ellipsometry-based Optical Scanner.

    Science.gov (United States)

    Landry, James P; Fei, Yiyan; Zhu, X D

    2011-12-01

    Small-molecule compounds remain the major source of therapeutic and preventative drugs. Developing new drugs against a protein target often requires screening large collections of compounds with diverse structures for ligands or ligand fragments that exhibit sufficiently affinity and desirable inhibition effect on the target before further optimization and development. Since the number of small molecule compounds is large, high-throughput screening (HTS) methods are needed. Small-molecule microarrays (SMM) on a solid support in combination with a suitable binding assay form a viable HTS platform. We demonstrate that by combining an oblique-incidence reflectivity difference optical scanner with SMM we can screen 10,000 small-molecule compounds on a single glass slide for protein ligands without fluorescence labeling. Furthermore using such a label-free assay platform we can simultaneously acquire binding curves of a solution-phase protein to over 10,000 immobilized compounds, thus enabling full characterization of protein-ligand interactions over a wide range of affinity constants.

  8. Homogeneous and label-free detection of microRNAs using bifunctional strand displacement amplification-mediated hyperbranched rolling circle amplification.

    Science.gov (United States)

    Zhang, Li-rong; Zhu, Guichi; Zhang, Chun-yang

    2014-07-01

    MicroRNAs (miRNAs) are an emerging class of biomarkers and therapeutic targets for various diseases including cancers. Here, we develop a homogeneous and label-free method for sensitive detection of let-7a miRNA based on bifunctional strand displacement amplification (SDA)-mediated hyperbranched rolling circle amplification (HRCA). The binding of target miRNA with the linear template initiates the bifunctional SDA reaction, generating two different kinds of triggers which can hybridize with the linear template to initiate new rounds of SDA reaction for the production of more and more triggers. In the meantime, the released two different kinds of triggers can function as the first and the second primers, respectively, to initiate the HRCA reaction whose products can be simply monitored by a standard fluorometer with SYBR Green I as the fluorescent indicator. The proposed method exhibits high sensitivity with a detection limit of as low as 1.8 × 10(-13) M and a large dynamic range of 5 orders of magnitude from 0.1 pM to 10 nM, and it can even discriminate the single-base difference among the miRNA family members. Moreover, this method can be used to analyze the total RNA samples from the human lung tissues and might be further applied for sensitive detection of various proteins, small molecules, and metal ions in combination with specific aptamers.

  9. Properties of targeted preamplification in DNA and cDNA quantification.

    Science.gov (United States)

    Andersson, Daniel; Akrap, Nina; Svec, David; Godfrey, Tony E; Kubista, Mikael; Landberg, Göran; Ståhlberg, Anders

    2015-01-01

    Quantification of small molecule numbers often requires preamplification to generate enough copies for accurate downstream enumerations. Here, we studied experimental parameters in targeted preamplification and their effects on downstream quantitative real-time PCR (qPCR). To evaluate different strategies, we monitored the preamplification reaction in real-time using SYBR Green detection chemistry followed by melting curve analysis. Furthermore, individual targets were evaluated by qPCR. The preamplification reaction performed best when a large number of primer pairs was included in the primer pool. In addition, preamplification efficiency, reproducibility and specificity were found to depend on the number of template molecules present, primer concentration, annealing time and annealing temperature. The amount of nonspecific PCR products could also be reduced about 1000-fold using bovine serum albumin, glycerol and formamide in the preamplification. On the basis of our findings, we provide recommendations how to perform robust and highly accurate targeted preamplification in combination with qPCR or next-generation sequencing.

  10. In Vitro Selection and Characterization of DNA Aptamers to a Small Molecule Target.

    Science.gov (United States)

    Ruscito, Annamaria; McConnell, Erin M; Koudrina, Anna; Velu, Ranganathan; Mattice, Christopher; Hunt, Vernon; McKeague, Maureen; DeRosa, Maria C

    2017-12-14

    Aptamers, synthetic oligonucleotide-based molecular recognition probes, have found use in a wide array of biosensing technologies based on their tight and highly selective binding to a variety of molecular targets. However, the inherent challenges associated with the selection and characterization of aptamers for small molecule targets have resulted in their underrepresentation, despite the need for small molecule detection in fields such as medicine, the environment, and agriculture. This protocol describes the steps in the selection, sequencing, affinity characterization, and truncation of DNA aptamers that are specific for small molecule targets. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

  11. Enhancing Targeted Genomic DNA Editing in Chicken Cells Using the CRISPR/Cas9 System

    Science.gov (United States)

    Wang, Ling; Yang, Likai; Guo, Yijie; Du, Weili; Yin, Yajun; Zhang, Tao; Lu, Hongzhao

    2017-01-01

    The CRISPR/Cas9 system has enabled highly efficient genome targeted editing for various organisms. However, few studies have focused on CRISPR/Cas9 nuclease-mediated chicken genome editing compared with mammalian genomes. The current study combined CRISPR with yeast Rad52 (yRad52) to enhance targeted genomic DNA editing in chicken DF-1 cells. The efficiency of CRISPR/Cas9 nuclease-induced targeted mutations in the chicken genome was increased to 41.9% via the enrichment of the dual-reporter surrogate system. In addition, the combined effect of CRISPR nuclease and yRad52 dramatically increased the efficiency of the targeted substitution in the myostatin gene using 50-mer oligodeoxynucleotides (ssODN) as the donor DNA, resulting in a 36.7% editing efficiency after puromycin selection. Furthermore, based on the effect of yRad52, the frequency of exogenous gene integration in the chicken genome was more than 3-fold higher than that without yRad52. Collectively, these results suggest that ssODN is an ideal donor DNA for targeted substitution and that CRISPR/Cas9 combined with yRad52 significantly enhances chicken genome editing. These findings could be extensively applied in other organisms. PMID:28068387

  12. A label-free and high sensitive aptamer biosensor based on hyperbranched polyester microspheres for thrombin detection

    International Nuclear Information System (INIS)

    Sun, Chong; Han, Qiaorong; Wang, Daoying; Xu, Weimin; Wang, Weijuan; Zhao, Wenbo; Zhou, Min

    2014-01-01

    Highlights: • A label-free thrombin aptamer biosensor applied in whole blood has been developed. • The aptamer biosensor showed a wide detection range and a low detection limit. • The antibiofouling idea utilized for biosensor is significant for diagnostics. - Abstract: In this paper, we have synthesized hyperbranched polyester microspheres with carboxylic acid functional groups (HBPE-CA) and developed a label-free electrochemical aptamer biosensor using thrombin-binding aptamer (TBA) as receptor for the measurement of thrombin in whole blood. The indium tin oxide (ITO) electrode surface modified with HBPE-CA microspheres was grafted with TBA, which has excellent binding affinity and selectivity for thrombin. Binding of the thrombin at the modified ITO electrode surface greatly restrained access of electrons for a redox probe of [Fe(CN) 6 ] 3−/4− . Moreover, the aptamer biosensor could be used for detection of thrombin in whole blood, a wide detection range (10 fM–100 nM) and a detection limit on the order of 0.90 fM were demonstrated. Control experiments were also carried out by using bull serum albumin (BSA) and lysozyme in the absence of thrombin. The good stability and repeatability of this aptamer biosensor were also proved. We expect that this demonstration will lead to the development of highly sensitive label-free sensors based on aptamer with lower cost than current technology. The integration of the technologies, which include anticoagulant, sensor and nanoscience, will bring significant input to high-performance biosensors relevant to diagnostics and therapy of interest for human health

  13. A label-free and high sensitive aptamer biosensor based on hyperbranched polyester microspheres for thrombin detection

    Energy Technology Data Exchange (ETDEWEB)

    Sun, Chong [Jiangsu Key Laboratory of Biofunctional Materials, Biomedical Functional Materials Collaborative Innovation Center, College of Chemistry and Materials Science, Nanjing Normal University, Nanjing 210023 (China); Institute of Agricultural Products Processing, Jiangsu Academy of Agricultural Sciences, Nanjing 210014 (China); Han, Qiaorong [Jiangsu Key Laboratory of Biofunctional Materials, Biomedical Functional Materials Collaborative Innovation Center, College of Chemistry and Materials Science, Nanjing Normal University, Nanjing 210023 (China); Wang, Daoying; Xu, Weimin [Institute of Agricultural Products Processing, Jiangsu Academy of Agricultural Sciences, Nanjing 210014 (China); Wang, Weijuan [Jiangsu Key Laboratory of Biofunctional Materials, Biomedical Functional Materials Collaborative Innovation Center, College of Chemistry and Materials Science, Nanjing Normal University, Nanjing 210023 (China); Zhao, Wenbo, E-mail: zhaowenbo@njnu.edu.cn [Jiangsu Key Laboratory of Biofunctional Materials, Biomedical Functional Materials Collaborative Innovation Center, College of Chemistry and Materials Science, Nanjing Normal University, Nanjing 210023 (China); Zhou, Min, E-mail: zhouminnju@126.com [Department of Vascular Surgery, the Affiliated Drum Tower Hospital of Nanjing University Medical School, Nanjing 210008 (China)

    2014-11-19

    Highlights: • A label-free thrombin aptamer biosensor applied in whole blood has been developed. • The aptamer biosensor showed a wide detection range and a low detection limit. • The antibiofouling idea utilized for biosensor is significant for diagnostics. - Abstract: In this paper, we have synthesized hyperbranched polyester microspheres with carboxylic acid functional groups (HBPE-CA) and developed a label-free electrochemical aptamer biosensor using thrombin-binding aptamer (TBA) as receptor for the measurement of thrombin in whole blood. The indium tin oxide (ITO) electrode surface modified with HBPE-CA microspheres was grafted with TBA, which has excellent binding affinity and selectivity for thrombin. Binding of the thrombin at the modified ITO electrode surface greatly restrained access of electrons for a redox probe of [Fe(CN){sub 6}]{sup 3−/4−}. Moreover, the aptamer biosensor could be used for detection of thrombin in whole blood, a wide detection range (10 fM–100 nM) and a detection limit on the order of 0.90 fM were demonstrated. Control experiments were also carried out by using bull serum albumin (BSA) and lysozyme in the absence of thrombin. The good stability and repeatability of this aptamer biosensor were also proved. We expect that this demonstration will lead to the development of highly sensitive label-free sensors based on aptamer with lower cost than current technology. The integration of the technologies, which include anticoagulant, sensor and nanoscience, will bring significant input to high-performance biosensors relevant to diagnostics and therapy of interest for human health.

  14. An MCMC Algorithm for Target Estimation in Real-Time DNA Microarrays

    Directory of Open Access Journals (Sweden)

    Vikalo Haris

    2010-01-01

    Full Text Available DNA microarrays detect the presence and quantify the amounts of nucleic acid molecules of interest. They rely on a chemical attraction between the target molecules and their Watson-Crick complements, which serve as biological sensing elements (probes. The attraction between these biomolecules leads to binding, in which probes capture target analytes. Recently developed real-time DNA microarrays are capable of observing kinetics of the binding process. They collect noisy measurements of the amount of captured molecules at discrete points in time. Molecular binding is a random process which, in this paper, is modeled by a stochastic differential equation. The target analyte quantification is posed as a parameter estimation problem, and solved using a Markov Chain Monte Carlo technique. In simulation studies where we test the robustness with respect to the measurement noise, the proposed technique significantly outperforms previously proposed methods. Moreover, the proposed approach is tested and verified on experimental data.

  15. Targeted DNA delivery to cancer cells using a biotinylated chitosan carrier.

    Science.gov (United States)

    Darvishi, Mohammad H; Nomani, Alireza; Hashemzadeh, Hadi; Amini, Mohsen; Shokrgozar, Mohammad A; Dinarvand, Rassoul

    2017-05-01

    A novel biotinylated chitosan-graft-polyethyleneimine (Bio-Chi-g-PEI) copolymer was synthesized and evaluated as a nonviral gene delivery carrier for improvement of the transfection efficiency, endosomal escape, and targeted gene delivery of a plasmid encoding green fluorescent protein N1 (pEGFP-N1) into two different biotin-overexpressing cell lines including HeLa and OVCAR-3 cells. The structure of the obtained copolymers was confirmed by 1 H nuclear magnetic resonance ( 1 H NMR) and Fourier transform infrared spectroscopy. Physicochemical properties of the Bio-Chi-g-PEI/plasmid DNA (pDNA) complexes such as complex stability, size, zeta potential, and their morphology were investigated at various weight ratios of copolymer to pDNA. Bio-Chi-g-PEI copolymers could effectively condense pDNA into small particles with average diameters less than 164 nm and the zeta potential of +34.8 mV at the N/P ratio of 40/1. As revealed by flow cytometry, Bio-Chi-g-PEI/pDNA complexes had lower cytotoxicity than that of PEI 25 kDa/pDNA complexes in both cell lines. In vitro experiments revealed that the Bio-Chi-gPEI/pDNA complexes not only had much lower cytotoxicity, but also displayed higher transfection efficiency than that of PEI 25kDa/pDNA complexes. High percentage of cancer cells was successfully transfected by Bio-Chi-g-PEI/pDNA and properly expressed GFP protein. This study indicates that this copolymer complex can be a promising gene delivery carrier. © 2016 International Union of Biochemistry and Molecular Biology, Inc.

  16. Single walled carbon nanotube-based electrical biosensor for the label-free detection of pathogenic bacteria

    DEFF Research Database (Denmark)

    Yoo, S. M.; Baek, Y. K.; Shin, S.

    2016-01-01

    We herein describe the development of a single-walled carbon nanotube (SWNT)-based electrical biosensor consisting of a two-terminal resistor, and report its use for the specific, label-free detection of pathogenic bacteria via changes in conductance. The ability of this biosensor to recognize...... different pathogenic bacteria was analyzed, and conditions were optimized with different probe concentrations. Using this system, the reference strains and clinical isolates of Staphylococcus aureus and Escherichia coli were successfully detected; in both cases, the sensor showed a detection limit of 10 CFU....... This SWNT-based electrical biosensor will prove useful for the development of highly sensitive and specific handheld pathogen detectors....

  17. Novel charge plasma based dielectric modulated impact ionization MOSFET as a biosensor for label-free detection

    Science.gov (United States)

    Chanda, Manash; Dey, Prithu; De, Swapnadip; Sarkar, Chandan Kumar

    2015-10-01

    In this paper a charge plasma based dielectric modulated impact ionization MOSFET (CP-DIMOSFET) has been proposed for the first time to ease the label free detection of biomolecules. The concept of CP-DIMOSFET is proposed and analyzed on basis of simulated data using SILVACO ATLAS. Low thermal budgeting and thin silicon layer without any dopant implantations make the proposed structure advantageous compared to the existing MOSFET based biosensors. The results show that the proposed device is capable to detect the presence of biomolecules. Simple fabrication schemes, miniaturization, high sensitivity, dominance of dielectric modulation make the proposed biosensor a promising one that could one day revolutionize the healthcare industry.

  18. Direct and label-free detection of the human growth hormone in urine by an ultrasensitive bimodal waveguide biosensor.

    Science.gov (United States)

    González-Guerrero, Ana Belén; Maldonado, Jesús; Dante, Stefania; Grajales, Daniel; Lechuga, Laura M

    2017-01-01

    A label-free interferometric transducer showing a theoretical detection limit for homogeneous sensing of 5 × 10 -8 RIU, being equivalent to a protein mass coverage resolution of 2.8 fg mm -2 , is used to develop a high sensitive biosensor for protein detection. The extreme sensitivity of this transducer combined with a selective bioreceptor layer enables the direct evaluation of the human growth hormone (hGH) in undiluted urine matrix in the 10 pg mL -1 range. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Quinone-Based Polymers for Label-Free and Reagentless Electrochemical Immunosensors: Application to Proteins, Antibodies and Pesticides Detection

    Directory of Open Access Journals (Sweden)

    Minh-Chau Pham

    2013-01-01

    Full Text Available Polyquinone derivatives are widely recognized in the literature for their remarkable properties, their biocompatibility, simple synthesis, and easy bio-functionalization. We have shown that polyquinones present very stable electroactivity in neutral aqueous medium within the cathodic potential domain avoiding side oxidation of interfering species. Besides, they can act as immobilized redox transducers for probing biomolecular interactions in sensors. Our group has been working on devices based on such modified electrodes with a view to applications for proteins, antibodies and organic pollutants using a reagentless label-free electrochemical immunosensor format. Herein, these developments are briefly reviewed and put into perspective.

  20. Long-circulating DNA lipid nanocapsules as new vector for passive tumor targeting.

    Science.gov (United States)

    Morille, Marie; Montier, Tristan; Legras, Pierre; Carmoy, Nathalie; Brodin, Priscille; Pitard, Bruno; Benoît, Jean-Pierre; Passirani, Catherine

    2010-01-01

    Systemic gene delivery systems are needed for therapeutic application to organs that are inaccessible by percutaneous injection. Currently, the main objective is the development of a stable and non-toxic vector that can encapsulate and deliver foreign genetic material to target cells. To this end, DNA, complexed with cationic lipids i.e. DOTAP/DOPE, was encapsulated into lipid nanocapsules (LNCs) leading to the formation of stable nanocarriers (DNA LNCs) with a size inferior to 130 nm. Amphiphilic and flexible poly (ethylene glycol) (PEG) polymer coatings [PEG lipid derivative (DSPE-mPEG(2000)) or F108 poloxamer] at different concentrations were selected to make DNA LNCs stealthy. Some of these coated lipid nanocapsules were able to inhibit complement activation and were not phagocytized in vitro by macrophagic THP-1 cells whereas uncoated DNA LNCs accumulated in the vacuolar compartment of THP-1 cells. These results correlated with a significant increase of in vivo circulation time in mice especially for DSPE-mPEG(2000) 10 mm and an early half-life time (t(1/2) of distribution) 5-fold greater than for non-coated DNA LNCs (7.1 h vs 1.4 h). Finally, a tumor accumulation assessed by in vivo fluorescence imaging system was evidenced for these coated LNCs as a passive targeting without causing any hepatic damage.

  1. Targeted DNA demethylation of the Arabidopsis genome using the human TET1 catalytic domain

    Science.gov (United States)

    Gallego-Bartolomé, Javier; Gardiner, Jason; Liu, Wanlu; Papikian, Ashot; Ghoshal, Basudev; Kuo, Hsuan Yu; Zhao, Jenny Miao-Chi; Jacobsen, Steven E.

    2018-01-01

    DNA methylation is an important epigenetic modification involved in gene regulation and transposable element silencing. Changes in DNA methylation can be heritable and, thus, can lead to the formation of stable epialleles. A well-characterized example of a stable epiallele in plants is fwa, which consists of the loss of DNA cytosine methylation (5mC) in the promoter of the FLOWERING WAGENINGEN (FWA) gene, causing up-regulation of FWA and a heritable late-flowering phenotype. Here we demonstrate that a fusion between the catalytic domain of the human demethylase TEN-ELEVEN TRANSLOCATION1 (TET1cd) and an artificial zinc finger (ZF) designed to target the FWA promoter can cause highly efficient targeted demethylation, FWA up-regulation, and a heritable late-flowering phenotype. Additional ZF–TET1cd fusions designed to target methylated regions of the CACTA1 transposon also caused targeted demethylation and changes in expression. Finally, we have developed a CRISPR/dCas9-based targeted demethylation system using the TET1cd and a modified SunTag system. Similar to the ZF–TET1cd fusions, the SunTag–TET1cd system is able to target demethylation and activate gene expression when directed to the FWA or CACTA1 loci. Our study provides tools for targeted removal of 5mC at specific loci in the genome with high specificity and minimal off-target effects. These tools provide the opportunity to develop new epialleles for traits of interest, and to reactivate expression of previously silenced genes, transgenes, or transposons. PMID:29444862

  2. Quantification of differential gene expression by multiplexed targeted resequencing of cDNA

    Science.gov (United States)

    Arts, Peer; van der Raadt, Jori; van Gestel, Sebastianus H.C.; Steehouwer, Marloes; Shendure, Jay; Hoischen, Alexander; Albers, Cornelis A.

    2017-01-01

    Whole-transcriptome or RNA sequencing (RNA-Seq) is a powerful and versatile tool for functional analysis of different types of RNA molecules, but sample reagent and sequencing cost can be prohibitive for hypothesis-driven studies where the aim is to quantify differential expression of a limited number of genes. Here we present an approach for quantification of differential mRNA expression by targeted resequencing of complementary DNA using single-molecule molecular inversion probes (cDNA-smMIPs) that enable highly multiplexed resequencing of cDNA target regions of ∼100 nucleotides and counting of individual molecules. We show that accurate estimates of differential expression can be obtained from molecule counts for hundreds of smMIPs per reaction and that smMIPs are also suitable for quantification of relative gene expression and allele-specific expression. Compared with low-coverage RNA-Seq and a hybridization-based targeted RNA-Seq method, cDNA-smMIPs are a cost-effective high-throughput tool for hypothesis-driven expression analysis in large numbers of genes (10 to 500) and samples (hundreds to thousands). PMID:28474677

  3. Simultaneous detection of multiple DNA targets by integrating dual-color graphene quantum dot nanoprobes and carbon nanotubes.

    Science.gov (United States)

    Qian, Zhaosheng; Shan, Xiaoyue; Chai, Lujing; Chen, Jianrong; Feng, Hui

    2014-12-01

    Simultaneous detection of multiple DNA targets was achieved based on a biocompatible graphene quantum dots (GQDs) and carbon nanotubes (CNTs) platform through spontaneous assembly between dual-color GQD-based probes and CNTs and subsequently self-recognition between DNA probes and targets. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Is DNA Alive? A Study of Conceptual Change Through Targeted Instruction

    Science.gov (United States)

    Witzig, Stephen B.; Freyermuth, Sharyn K.; Siegel, Marcelle A.; Izci, Kemal; Pires, J. Chris

    2013-08-01

    We are involved in a project to incorporate innovative assessments within a reform-based large-lecture biochemistry course for nonmajors. We not only assessed misconceptions but purposefully changed instruction throughout the semester to confront student ideas. Our research questions targeted student conceptions of deoxyribonucleic acid (DNA) along with understanding in what ways classroom discussions/activities influence student conceptions. Data sources included pre-/post-assessments, semi-structured interviews, and student work on exams/assessments. We found that students held misconceptions about the chemical nature of DNA, with 63 % of students claiming that DNA is alive prior to instruction. The chemical nature of DNA is an important fundamental concept in science fields. We confronted this misconception throughout the semester collecting data from several instructional interventions. Case studies of individual students revealed how various instructional strategies/assessments allowed students to construct and demonstrate the scientifically accepted understanding of the chemical nature of DNA. However, the post-assessment exposed that 40 % of students still held misconceptions about DNA, indicating the persistent nature of this misconception. Implications for teaching and learning are discussed.

  5. Antibacterial small molecules targeting the conserved TOPRIM domain of DNA gyrase.

    Directory of Open Access Journals (Sweden)

    Scott S Walker

    Full Text Available To combat the threat of antibiotic-resistant Gram-negative bacteria, novel agents that circumvent established resistance mechanisms are urgently needed. Our approach was to focus first on identifying bioactive small molecules followed by chemical lead prioritization and target identification. Within this annotated library of bioactives, we identified a small molecule with activity against efflux-deficient Escherichia coli and other sensitized Gram-negatives. Further studies suggested that this compound inhibited DNA replication and selection for resistance identified mutations in a subunit of E. coli DNA gyrase, a type II topoisomerase. Our initial compound demonstrated weak inhibition of DNA gyrase activity while optimized compounds demonstrated significantly improved inhibition of E. coli and Pseudomonas aeruginosa DNA gyrase and caused cleaved complex stabilization, a hallmark of certain bactericidal DNA gyrase inhibitors. Amino acid substitutions conferring resistance to this new class of DNA gyrase inhibitors reside exclusively in the TOPRIM domain of GyrB and are not associated with resistance to the fluoroquinolones, suggesting a novel binding site for a gyrase inhibitor.

  6. DNA Immobilization and Hybridization Detection by the Intrinsic Molecular Charge Using Capacitive Field-Effect Sensors Modified with a Charged Weak Polyelectrolyte Layer.

    Science.gov (United States)

    Bronder, Thomas S; Poghossian, Arshak; Scheja, Sabrina; Wu, Chunsheng; Keusgen, Michael; Mewes, Dieter; Schöning, Michael J

    2015-09-16

    Miniaturized setup, compatibility with advanced micro- and nanotechnologies, and ability to detect biomolecules by their intrinsic molecular charge favor the semiconductor field-effect platform as one of the most attractive approaches for the development of label-free DNA chips. In this work, a capacitive field-effect EIS (electrolyte-insulator-semiconductor) sensor covered with a layer-by-layer prepared, positively charged weak polyelectrolyte layer of PAH (poly(allylamine hydrochloride)) was used for the label-free electrical detection of DNA (deoxyribonucleic acid) immobilization and hybridization. The negatively charged probe single-stranded DNA (ssDNA) molecules were electrostatically adsorbed onto the positively charged PAH layer, resulting in a preferentially flat orientation of the ssDNA molecules within the Debye length, thus yielding a reduced charge-screening effect and a higher sensor signal. Each sensor-surface modification step (PAH adsorption, probe ssDNA immobilization, hybridization with complementary target DNA (cDNA), reducing an unspecific adsorption by a blocking agent, incubation with noncomplementary DNA (ncDNA) solution) was monitored by means of capacitance-voltage and constant-capacitance measurements. In addition, the surface morphology of the PAH layer was studied by atomic force microscopy and contact-angle measurements. High hybridization signals of 34 and 43 mV were recorded in low-ionic strength solutions of 10 and 1 mM, respectively. In contrast, a small signal of 4 mV was recorded in the case of unspecific adsorption of fully mismatched ncDNA. The density of probe ssDNA and dsDNA molecules as well as the hybridization efficiency was estimated using the experimentally measured DNA immobilization and hybridization signals and a simplified double-layer capacitor model. The results of field-effect experiments were supported by fluorescence measurements, verifying the DNA-immobilization and hybridization event.

  7. Indium-tin-oxide thin film transistor biosensors for label-free detection of avian influenza virus H5N1

    International Nuclear Information System (INIS)

    Guo, Di; Zhuo, Ming; Zhang, Xiaoai; Xu, Cheng; Jiang, Jie; Gao, Fu; Wan, Qing; Li, Qiuhong; Wang, Taihong

    2013-01-01

    Highlights: ► A highly selective label-free biosensor is established based on indium-tin-oxide thin-film transistors (ITO TFTs). ► AI H5N1 virus was successfully detected through shift in threshold voltage and field-effect mobility of ITO TFT. ► The ITO TFT is applied in biosensor for the first time and shows good reusability and stability. ► Fabrication of the platform is simple with low cost, which is suitable for mass commercial production. -- Abstract: As continuous outbreak of avian influenza (AI) has become a threat to human health, economic development and social stability, it is urgently necessary to detect the highly pathogenic avian influenza H5N1 virus quickly. In this study, we fabricated indium-tin-oxide thin-film transistors (ITO TFTs) on a glass substrate for the detecting of AI H5N1. The ITO TFT is fabricated by a one-shadow-mask process in which a channel layer can be simultaneously self-assembled between ITO source/drain electrodes during magnetron sputtering deposition. Monoclonal anti-H5N1 antibodies specific for AI H5N1 virus were covalently immobilized on the ITO channel by (3-glycidoxypropyl)trimethoxysilane. The introduction of target AI H5N1 virus affected the electronic properties of the ITO TFT, which caused a change in the resultant threshold voltage (V T ) and field-effect mobility. The changes of I D –V G curves were consistent with an n-type field effect transistor behavior affected by nearby negatively charged AI H5N1 viruses. The transistor based sensor demonstrated high selectivity and stability for AI H5N1 virus sensing. The sensor showed linear response to AI H5N1 in the concentrations range from 5 × 10 −9 g mL −1 to 5 × 10 −6 g mL −1 with a detection limit of 0.8 × 10 −10 g mL −1 . Moreover, the ITO TFT biosensors can be repeatedly used through the washing processes. With its excellent electric properties and the potential for mass commercial production, ITO TFTs can be promising candidates for the

  8. Indium-tin-oxide thin film transistor biosensors for label-free detection of avian influenza virus H5N1

    Energy Technology Data Exchange (ETDEWEB)

    Guo, Di; Zhuo, Ming [Key Laboratory for Micro-Nano Optoelectronic Devices of Ministry of Education, State Key Laboratory for Chemo/Biosensing and Chemometrics, Hunan University, Changsha 410082 (China); Zhang, Xiaoai [State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing (China); Xu, Cheng; Jiang, Jie [Key Laboratory for Micro-Nano Optoelectronic Devices of Ministry of Education, State Key Laboratory for Chemo/Biosensing and Chemometrics, Hunan University, Changsha 410082 (China); Gao, Fu [State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing (China); Wan, Qing, E-mail: wanqing7686@hotmail.com [Key Laboratory for Micro-Nano Optoelectronic Devices of Ministry of Education, State Key Laboratory for Chemo/Biosensing and Chemometrics, Hunan University, Changsha 410082 (China); Li, Qiuhong, E-mail: liqiuhong2004@hotmail.com [Key Laboratory for Micro-Nano Optoelectronic Devices of Ministry of Education, State Key Laboratory for Chemo/Biosensing and Chemometrics, Hunan University, Changsha 410082 (China); Wang, Taihong, E-mail: thwang@hnu.cn [Key Laboratory for Micro-Nano Optoelectronic Devices of Ministry of Education, State Key Laboratory for Chemo/Biosensing and Chemometrics, Hunan University, Changsha 410082 (China)

    2013-04-22

    Highlights: ► A highly selective label-free biosensor is established based on indium-tin-oxide thin-film transistors (ITO TFTs). ► AI H5N1 virus was successfully detected through shift in threshold voltage and field-effect mobility of ITO TFT. ► The ITO TFT is applied in biosensor for the first time and shows good reusability and stability. ► Fabrication of the platform is simple with low cost, which is suitable for mass commercial production. -- Abstract: As continuous outbreak of avian influenza (AI) has become a threat to human health, economic development and social stability, it is urgently necessary to detect the highly pathogenic avian influenza H5N1 virus quickly. In this study, we fabricated indium-tin-oxide thin-film transistors (ITO TFTs) on a glass substrate for the detecting of AI H5N1. The ITO TFT is fabricated by a one-shadow-mask process in which a channel layer can be simultaneously self-assembled between ITO source/drain electrodes during magnetron sputtering deposition. Monoclonal anti-H5N1 antibodies specific for AI H5N1 virus were covalently immobilized on the ITO channel by (3-glycidoxypropyl)trimethoxysilane. The introduction of target AI H5N1 virus affected the electronic properties of the ITO TFT, which caused a change in the resultant threshold voltage (V{sub T}) and field-effect mobility. The changes of I{sub D}–V{sub G} curves were consistent with an n-type field effect transistor behavior affected by nearby negatively charged AI H5N1 viruses. The transistor based sensor demonstrated high selectivity and stability for AI H5N1 virus sensing. The sensor showed linear response to AI H5N1 in the concentrations range from 5 × 10{sup −9} g mL{sup −1} to 5 × 10{sup −6} g mL{sup −1} with a detection limit of 0.8 × 10{sup −10} g mL{sup −1}. Moreover, the ITO TFT biosensors can be repeatedly used through the washing processes. With its excellent electric properties and the potential for mass commercial production, ITO TFTs

  9. Multiway study of hybridization in nanoscale semiconductor labeled DNA based on fluorescence resonance energy transfer

    DEFF Research Database (Denmark)

    Gholami, Somayeh; Kompany Zare, Mohsen

    2013-01-01

    donor-QD acceptor) upon hybridization with a label free target was monitored by two-dimensional photoluminescence excitation spectroscopy (2D-PLE). Detection of a target oligonucleotide strand, using sandwiched nanoassembly in a separation-free format, was performed with the appearance of a new feature...... and model based analysis of 2D-PLE data was implemented by means of PAR-AFAC and hard trilinear decomposition (HTD), allowing to fit a proper model for FRET-based sandwich DNA hybridization systems. This study is the first successful application of a multiway chemometric technique to consider FRET based DNA...... hybridization in sandwiched nanoassemblies. A multi-equilibria model was properly fitted to the data and confirmed there is a competition between ternary and binary complex formation. Equilibrium constants of DNA hybridization in sandwiched nanoassemblies were estimated for the first time. Equilibrium constants...

  10. Programmable type III-A CRISPR-Cas DNA targeting modules.

    Directory of Open Access Journals (Sweden)

    H Travis Ichikawa

    Full Text Available The CRISPR-Cas systems provide invader defense in a wide variety of prokaryotes, as well as technologies for many powerful applications. The Type III-A or Csm CRISPR-Cas system is one of the most widely distributed across prokaryotic phyla, and cleaves targeted DNA and RNA molecules. In this work, we have constructed modules of Csm systems from 3 bacterial species and heterologously expressed the functional modules in E. coli. The modules include a Cas6 protein and a CRISPR locus for crRNA production, and Csm effector complex proteins. The expressed modules from L. lactis, S. epidermidis and S. thermophilus specifically eliminate invading plasmids recognized by the crRNAs of the systems. Characteristically, activation of plasmid targeting activity depends on transcription of the plasmid sequence recognized by the crRNA. Activity was not observed when transcription of the crRNA target sequence was blocked, or when the opposite strand or a non-target sequence was transcribed. Moreover, the Csm module can be programmed to recognize plasmids with novel target sequences by addition of appropriate crRNA coding sequences to the module. These systems provide a platform for investigation of Type III-A CRISPR-Cas systems in E. coli, and for introduction of programmable transcription-activated DNA targeting into novel organisms.

  11. Spectral-domain optical coherence phase microscopy for label-free multiplexed protein microarray assay

    NARCIS (Netherlands)

    Joo, C.; Ozkumur, E.; Unlu, B.; de Boer, J.F.

    2009-01-01

    Quantitative measurement of affinities and kinetics of various biomolecular interactions such as protein-protein, protein-DNA and receptor-ligand is central to our understanding of basic molecular and cellular functions and is useful for therapeutic evaluation. Here, we describe a laser-scanning

  12. Label-free imaging of the native, living cellular nanoarchitecture using partial-wave spectroscopic microscopy

    DEFF Research Database (Denmark)

    Almassalha, Luay M; Bauer, Greta M; Chandler, John E

    2016-01-01

    The organization of chromatin is a regulator of molecular processes including transcription, replication, and DNA repair. The structures within chromatin that regulate these processes span from the nucleosomal (10-nm) to the chromosomal (>200-nm) levels, with little known about the dynamics of ch...

  13. Twin target self-amplification-based DNA machine for highly sensitive detection of cancer-related gene.

    Science.gov (United States)

    Xu, Huo; Jiang, Yifan; Liu, Dengyou; Liu, Kai; Zhang, Yafeng; Yu, Suhong; Shen, Zhifa; Wu, Zai-Sheng

    2018-06-29

    The sensitive detection of cancer-related genes is of great significance for early diagnosis and treatment of human cancers, and previous isothermal amplification sensing systems were often based on the reuse of target DNA, the amplification of enzymatic products and the accumulation of reporting probes. However, no reporting probes are able to be transformed into target species and in turn initiate the signal of other probes. Herein we reported a simple, isothermal and highly sensitive homogeneous assay system for tumor suppressor p53 gene detection based on a new autonomous DNA machine, where the signaling probe, molecular beacon (MB), was able to execute the function similar to target DNA besides providing the common signal. In the presence of target p53 gene, the operation of DNA machine can be initiated, and cyclical nucleic acid strand-displacement polymerization (CNDP) and nicking/polymerization cyclical amplification (NPCA) occur, during which the MB was opened by target species and cleaved by restriction endonuclease. In turn, the cleaved fragments could activate the next signaling process as target DNA did. According to the functional similarity, the cleaved fragment was called twin target, and the corresponding fashion to amplify the signal was named twin target self-amplification. Utilizing this newly-proposed DNA machine, the target DNA could be detected down to 0.1 pM with a wide dynamic range (6 orders of magnitude) and single-base mismatched targets were discriminated, indicating a very high assay sensitivity and good specificity. In addition, the DNA machine was not only used to screen the p53 gene in complex biological matrix but also was capable of practically detecting genomic DNA p53 extracted from A549 cell line. This indicates that the proposed DNA machine holds the potential application in biomedical research and early clinical diagnosis. Copyright © 2018 Elsevier B.V. All rights reserved.

  14. The Cytotoxicity Mechanism of 6-Shogaol-Treated HeLa Human Cervical Cancer Cells Revealed by Label-Free Shotgun Proteomics and Bioinformatics Analysis

    Directory of Open Access Journals (Sweden)

    Qun Liu

    2012-01-01

    Full Text Available Cervical cancer is one of the most common cancers among women in the world. 6-Shogaol is a natural compound isolated from the rhizome of ginger (Zingiber officinale. In this paper, we demonstrated that 6-shogaol induced apoptosis and G2/M phase arrest in human cervical cancer HeLa cells. Endoplasmic reticulum stress and mitochondrial pathway were involved in 6-shogaol-mediated apoptosis. Proteomic analysis based on label-free strategy by liquid chromatography chip quadrupole time-of-flight mass spectrometry was subsequently proposed to identify, in a non-target-biased manner, the molecular changes in cellular proteins in response to 6-shogaol treatment. A total of 287 proteins were differentially expressed in response to 24 h treatment with 15 μM 6-shogaol in HeLa cells. Significantly changed proteins were subjected to functional pathway analysis by multiple analyzing software. Ingenuity pathway analysis (IPA suggested that 14-3-3 signaling is a predominant canonical pathway involved in networks which may be significantly associated with the process of apoptosis and G2/M cell cycle arrest induced by 6-shogaol. In conclusion, this work developed an unbiased protein analysis strategy by shotgun proteomics and bioinformatics analysis. Data observed provide a comprehensive analysis of the 6-shogaol-treated HeLa cell proteome and reveal protein alterations that are associated with its anticancer mechanism.

  15. A flexible statistical model for alignment of label-free proteomics data--incorporating ion mobility and product ion information.

    Science.gov (United States)

    Benjamin, Ashlee M; Thompson, J Will; Soderblom, Erik J; Geromanos, Scott J; Henao, Ricardo; Kraus, Virginia B; Moseley, M Arthur; Lucas, Joseph E

    2013-12-16

    The goal of many proteomics experiments is to determine the abundance of proteins in biological samples, and the variation thereof in various physiological conditions. High-throughput quantitative proteomics, specifically label-free LC-MS/MS, allows rapid measurement of thousands of proteins, enabling large-scale studies of various biological systems. Prior to analyzing these information-rich datasets, raw data must undergo several computational processing steps. We present a method to address one of the essential steps in proteomics data processing--the matching of peptide measurements across samples. We describe a novel method for label-free proteomics data alignment with the ability to incorporate previously unused aspects of the data, particularly ion mobility drift times and product ion information. We compare the results of our alignment method to PEPPeR and OpenMS, and compare alignment accuracy achieved by different versions of our method utilizing various data characteristics. Our method results in increased match recall rates and similar or improved mismatch rates compared to PEPPeR and OpenMS feature-based alignment. We also show that the inclusion of drift time and product ion information results in higher recall rates and more confident matches, without increases in error rates. Based on the results presented here, we argue that the incorporation of ion mobility drift time and product ion information are worthy pursuits. Alignment methods should be flexible enough to utilize all available data, particularly with recent advancements in experimental separation methods.

  16. A SIMULTANEOUS MULTI-PROBE DETECTION LABEL-FREE OPTICAL-RESOLUTION PHOTOACOUSTIC MICROSCOPY TECHNIQUE BASED ON MICROCAVITY TRANSDUCER

    Directory of Open Access Journals (Sweden)

    YONGBO WU

    2013-07-01

    Full Text Available We demonstrate the feasibility of simultaneous multi-probe detection for an optical-resolution photoacoustic microscopy (OR-PAM system. OR-PAM has elicited the attention of biomedical imaging researchers because of its optical absorption contrast and high spatial resolution with great imaging depth. OR-PAM allows label-free and noninvasive imaging by maximizing the optical absorption of endogenous biomolecules. However, given the inadequate absorption of some biomolecules, detection sensitivity at the same incident intensity requires improvement. In this study, a modulated continuous wave with power density less than 3 mW/cm2 (1/4 of the ANSI safety limit excited the weak photoacoustic (PA signals of biological cells. A microcavity transducer is developed based on the bulk modulus of gas five orders of magnitude lower than that of solid; air pressure variation is inversely proportional to cavity volume at the same temperature increase. Considering that a PA wave expands in various directions, detecting PA signals from different positions and adding them together can increase detection sensitivity and signal-to-noise ratio. Therefore, we employ four detectors to acquire tiny PA signals simultaneously. Experimental results show that the developed OR-PAM system allows the label-free imaging of cells with weak optical absorption.

  17. Label-free detection of cellular drug responses by high-throughput bright-field imaging and machine learning.

    Science.gov (United States)

    Kobayashi, Hirofumi; Lei, Cheng; Wu, Yi; Mao, Ailin; Jiang, Yiyue; Guo, Baoshan; Ozeki, Yasuyuki; Goda, Keisuke

    2017-09-29

    In the last decade, high-content screening based on multivariate single-cell imaging has been proven effective in drug discovery to evaluate drug-induced phenotypic variations. Unfortunately, this method inherently requires fluorescent labeling which has several drawbacks. Here we present a label-free method for evaluating cellular drug responses only by high-throughput bright-field imaging with the aid of machine learning algorithms. Specifically, we performed high-throughput bright-field imaging of numerous drug-treated and -untreated cells (N = ~240,000) by optofluidic time-stretch microscopy with high throughput up to 10,000 cells/s and applied machine learning to the cell images to identify their morphological variations which are too subtle for human eyes to detect. Consequently, we achieved a high accuracy of 92% in distinguishing drug-treated and -untreated cells without the need for labeling. Furthermore, we also demonstrated that dose-dependent, drug-induced morphological change from different experiments can be inferred from the classification accuracy of a single classification model. Our work lays the groundwork for label-free drug screening in pharmaceutical science and industry.

  18. Comparison of one- and two-dimensional liquid chromatography approaches in the label-free quantitative analysis of Methylocella silvestris.

    Science.gov (United States)

    Patel, Nisha A; Crombie, Andrew; Slade, Susan E; Thalassinos, Konstantinos; Hughes, Chris; Connolly, Joanne B; Langridge, James; Murrell, J Colin; Scrivens, James H

    2012-09-07

    The proteome of the bacterium Methylocella silvestris has been characterized using reversed phase ultra high pressure liquid chromatography (UPLC) and two-dimensional reversed phase (high pH)-reversed phase (low pH) UPLC prior to mass spectrometric analysis. Variations in protein expression levels were identified with the aid of label-free quantification in a study of soluble protein extracts from the organism grown using methane, succinate, or propane as a substrate. The number of first dimensional fractionation steps has been varied for 2D analyses, and the impact on data throughput and quality has been demonstrated. Comparisons have been made regarding required experimental considerations including total loading of biological samples required, instrument time, and resulting data file sizes. The data obtained have been evaluated with respect to number of protein identifications, confidence of assignments, sequence coverage, relative levels of proteins, and dynamic range. Good qualitative and quantitative agreement was observed between the different approaches, and the potential benefits and limitations of the reversed phase-reversed phase UPLC technique in label-free analysis are discussed. A preliminary screen of the protein regulation data has also been performed, providing evidence for a possible propane assimilation route.

  19. Label-free evaluation of hepatic microvesicular steatosis with multimodal coherent anti-Stokes Raman scattering microscopy.

    Directory of Open Access Journals (Sweden)

    Thuc T Le

    Full Text Available Hepatic microvesicular steatosis is a hallmark of drug-induced hepatotoxicity and early-stage fatty liver disease. Current histopathology techniques are inadequate for the clinical evaluation of hepatic microvesicular steatosis. In this paper, we explore the use of multimodal coherent anti-Stokes Raman scattering (CARS microscopy for the detection and characterization of hepatic microvesicular steatosis. We show that CARS microscopy is more sensitive than Oil Red O histology for the detection of microvesicular steatosis. Computer-assisted analysis of liver lipid level based on CARS signal intensity is consistent with triglyceride measurement using a standard biochemical assay. Most importantly, in a single measurement procedure on unprocessed and unstained liver tissues, multimodal CARS imaging provides a wealth of critical information including the detection of microvesicular steatosis and quantitation of liver lipid content, number and size of lipid droplets, and lipid unsaturation and packing order of lipid droplets. Such information can only be assessed by multiple different methods on processed and stained liver tissues or tissue extracts using current standard analytical techniques. Multimodal CARS microscopy also permits label-free identification of lipid-rich non-parenchymal cells. In addition, label-free and non-perturbative CARS imaging allow rapid screening of mitochondrial toxins-induced microvesicular steatosis in primary hepatocyte cultures. With its sensitivity and versatility, multimodal CARS microscopy should be a powerful tool for the clinical evaluation of hepatic microvesicular steatosis.

  20. Label-free aptamer-based sensor for specific detection of malathion residues by surface-enhanced Raman scattering

    Science.gov (United States)

    Nie, Yonghui; Teng, Yuanjie; Li, Pan; Liu, Wenhan; Shi, Qianwei; Zhang, Yuchao

    2018-02-01

    A novel label-free aptamer surface-enhanced Raman scattering (SERS) sensor for trace malathion residue detection was proposed. In this process, the binding of malathion molecule with aptamer is identified directly. The silver nanoparticles modified with positively charged spermine served as enhancing and capture reagents for the negatively charged aptamer. Then, the silver nanoparticles modified by aptamer were used to specifically capture the malathion. The SERS background spectra of spermine, aptamer, and malathion were recorded and distinguished with the spectrum of malathion-aptamer. To enhance the characteristic peak signal of malathion captured by the aptamer, the aggregate reagents (NaCl, KCl, MgCl2) were compared and selected. The selectivity of this method was verified in the mixed-pesticide standard solution, which included malathion, phosmet, chlorpyrifos-methyl, and fethion. Results show that malathion can be specifically identified when the mixed-pesticide interferences existed. The standard curve was established, presenting a good linear range of 5 × 10- 7 to 1 × 10- 5 mol·L- 1. The spiked experiments for tap water show good recoveries from 87.4% to 110.5% with a relative standard deviation of less than 4.22%. Therefore, the proposed label-free aptamer SERS sensor is convenient, specifically detects trace malathion residues, and can be applied for qualitative and quantitative analysis of other pesticides.

  1. Label and label-free based surface-enhanced Raman scattering for pathogen bacteria detection: A review.

    Science.gov (United States)

    Liu, Yu; Zhou, Haibo; Hu, Ziwei; Yu, Guangxia; Yang, Danting; Zhao, Jinshun

    2017-08-15

    Rapid, accurate detection of pathogen bacteria is a highly topical research area for the sake of food safety and public health. Surface-enhanced Raman scattering (SERS) is being considered as a powerful and attractive technique for pathogen bacteria detection, due to its sensitivity, high speed, comparatively low cost, multiplexing ability and portability. This contribution aims to give a comprehensive overview of SERS as a technique for rapid detection of pathogen bacteria based on label and label-free strategies. A brief tutorial on SERS is given first of all. Then we summarize the recent trends and developments of label and label-free based SERS applied to detection of pathogen bacteria, including the relatively complete interpretation of SERS spectra. In addition, multifunctional SERS platforms for pathogen bacteria in matrix are discussed as well. Furthermore, an outlook of the work done and a perspective on the future directions of SERS as a reliable tool for real-time pathogen bacteria detection are given. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. Multimodal label-free ex vivo imaging using a dual-wavelength microscope with axial chromatic aberration compensation.

    Science.gov (United States)

    Filippi, Andrea; Dal Sasso, Eleonora; Iop, Laura; Armani, Andrea; Gintoli, Michele; Sandri, Marco; Gerosa, Gino; Romanato, Filippo; Borile, Giulia

    2018-03-01

    Label-free microscopy is a very powerful technique that can be applied to study samples with no need for exogenous fluorescent probes, keeping the main benefits of multiphoton microscopy, such as longer penetration depths and intrinsic optical sectioning while enabling serial multitechniques examinations on the same specimen. Among the many label-free microscopy methods, harmonic generation (HG) is one of the most intriguing methods due to its generally low photo-toxicity and relative ease of implementation. Today, HG and common two-photon microscopy (TPM) are well-established techniques, and are routinely used in several research fields. However, they require a significant amount of fine-tuning to be fully exploited, making them quite difficult to perform in parallel. Here, we present our designed multimodal microscope, capable of performing simultaneously TPM and HG without any kind of compromise thanks to two, separate, individually optimized laser sources with axial chromatic aberration compensation. We also apply our setup to the examination of a plethora of ex vivo samples to prove its capabilities and the significant advantages of a multimodal approach. (2018) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE).

  3. A label-free colorimetric sensor for Pb2+ detection based on the acceleration of gold leaching by graphene oxide.

    Science.gov (United States)

    Shi, Xinhao; Gu, Wei; Zhang, Cuiling; Zhao, Longyun; Peng, Weidong; Xian, Yuezhong

    2015-03-14

    In this work, we developed a novel, label-free, colorimetric sensor for Pb(2+) detection based on the acceleration of gold leaching by graphene oxide (GO) at room temperature. Gold nanoparticles (AuNPs) can be dissolved in a thiosulfate (S2O3(2-)) aqueous environment in the presence of oxygen; however, the leaching rate is very slow due to the high activation energy (27.99 kJ mol(-1)). In order to enhance the reaction rate, some accelerators should be added. In comparison with the traditional accelerators (metal ions or middle ligands), we found that GO could efficiently accelerate the gold leaching reaction. Kinetic data demonstrate that the dissolution rate of gold in the Pb(2+)-S2O3(2-)-GO system is 5 times faster than that without GO at room temperature. In addition, the effects of surface modification and the nanoparticle size on the etching of AuNPs were investigated. Based on the GO-accelerated concentration-dependent colour changes of AuNPs, a colorimetric sensor for Pb(2+) detection was developed with a linear range from 0.1 to 20 μM and the limit of detection (LOD) was evaluated to be 0.05 μM. This colorimetric assay is simple, low-cost, label-free, and has numerous potential applications in the field of environmental chemistry.

  4. Exploration of two-dimensional bio-functionalized phosphorene nanosheets (black phosphorous) for label free haptoglobin electro-immunosensing applications

    Science.gov (United States)

    Tuteja, Satish K.; Neethirajan, Suresh

    2018-04-01

    We report on the development of an antibody-functionalized interface based on electrochemically active liquid-exfoliated two-dimensional phosphorene (Ph) nanosheets—also known as black phosphorous nanosheets—for the label-free electrochemical immunosensing of a haptoglobin (Hp) biomarker, a clinical marker of severe inflammation. The electrodeposition has been achieved over the screen-printed electrode (SPE) using liquid-assisted ultrasonically exfoliated black phosphorus nanosheets. Subsequently, Ph-SPEs bioconjugated with Hp antibodies (Ab), using electrostatic interactions via a poly-L-lysine linker for biointerface development. Electrochemical analysis demonstrates that the Ab-modified Ph-SPEs (Ab@Ph-SPE) exhibit enhanced electroconducting behavior as compared to the pristine electrodes. This Ab-functionalized phosphorene-based electrochemical immunosensor platform has demonstrated remarkable sensitivity and specificity, having a dynamic linear response range from 0.01-10 mg ml-1 for Hp in standard and serum samples with a low detection limit (˜0.011 mg ml-1) using the label-free electrochemical technique. The sensor electrodes were also studied with other closely relative interferents to investigate cross reactivity and specificity. This strategy opens up avenues to POC (point-of-care) and on-farm livestock disease monitoring technologies for multiplexed diagnosis in complex biological samples such as serum. The technique is simple in fabrication and provides an analytical response in less than 60 s.

  5. Classification of M1/M2-polarized human macrophages by label-free hyperspectral reflectance confocal microscopy and multivariate analysis.

    Science.gov (United States)

    Bertani, Francesca R; Mozetic, Pamela; Fioramonti, Marco; Iuliani, Michele; Ribelli, Giulia; Pantano, Francesco; Santini, Daniele; Tonini, Giuseppe; Trombetta, Marcella; Businaro, Luca; Selci, Stefano; Rainer, Alberto

    2017-08-21

    The possibility of detecting and classifying living cells in a label-free and non-invasive manner holds significant theranostic potential. In this work, Hyperspectral Imaging (HSI) has been successfully applied to the analysis of macrophagic polarization, given its central role in several pathological settings, including the regulation of tumour microenvironment. Human monocyte derived macrophages have been investigated using hyperspectral reflectance confocal microscopy, and hyperspectral datasets have been analysed in terms of M1 vs. M2 polarization by Principal Components Analysis (PCA). Following PCA, Linear Discriminant Analysis has been implemented for semi-automatic classification of macrophagic polarization from HSI data. Our results confirm the possibility to perform single-cell-level in vitro classification of M1 vs. M2 macrophages in a non-invasive and label-free manner with a high accuracy (above 98% for cells deriving from the same donor), supporting the idea of applying the technique to the study of complex interacting cellular systems, such in the case of tumour-immunity in vitro models.

  6. Description of an Advantageous Optical Label-Free Biosensing Interferometric Read-Out Method to Measure Biological Species

    Directory of Open Access Journals (Sweden)

    Miguel Holgado

    2014-02-01

    Full Text Available In this article we report a new, simple, and reliable optical read-out detection method able to assess Rotavirus present in human sera as well as in the viral pollution sources. It is based on the interference of two interferometers used as biophotonic transducers. The method significantly improves the optical label-free biosensing response measuring both, the concentration of the AgR and its corresponding size. Two different immunoassays were carried out: Bovine Serum Albumin (BSA, and the recognition by its antibody (anti-BSA; and Rotavirus (AgR and the recognition by its antibody (anti-AgR. In the cases studied, and using as model interferometer a simple Fabry-Perot transducer, we demonstrate a biosensing enhancement of two orders of magnitude in the Limit of Detection (LoD. In fact, this read-out optical method may have significant implications to enhance other optical label-free photonic transducers reported in the scientific literature.

  7. Label-free detection of HIV-1 infected cells via integration of optical tweezers and photoluminescence spectroscopy

    Science.gov (United States)

    Lugongolo, Masixole Yvonne; Ombinda-Lemboumba, Saturnin; Noto, Luyanda Lunga; Maaza, Malik; Mthunzi-Kufa, Patience

    2018-02-01

    The human immunodeficiency virus-1 (HIV-1) is currently detected using conventional qualitative and quantitative tests to determine the presence or absence of HIV in blood samples. However, the approach of these tests detects the presence of either viral antibodies or viral RNA that require labelling which may be costly, sophisticated and time consuming. A label-free approach of detecting the presence of HIV is therefore desirable. Of note optical tweezers can be coupled with other technologies including spectroscopy, which also investigates light-matter interactions. For example, coupling of optical tweezers with luminescence spectroscopy techniques has emerged as a powerful tool in biology for micro-manipulation, detection and analysis of individual cells. Integration of optical techniques has enabled studying biological particles in a label-free manner, whilst detecting functional groups and other essential molecules within mixed populations of cells. In the current study, an optical trapping system coupled to luminescence spectroscopy was utilised to detect the presence of HIV infection in TZM-bl cells in vitro. This was performed by infecting TZM-bl cells with the ZM53 HIV-1 pseudovirus, and incubating them for 48 hours prior analysis. The differences between infected and uninfected cells were thereafter displayed as shown by the spectrographs obtained. Combination of these two techniques has a potential in the field of infectious disease diagnostics.

  8. Application of confocal Raman micro-spectroscopy for label-free monitoring of oxidative stress in living bronchial cells

    Science.gov (United States)

    Surmacki, Jakub M.; Quirós Gonzalez, Isabel; Bohndiek, Sarah E.

    2018-02-01

    Oxidative stress in cancer is implicated in tumor progression, being associated with increased therapy resistance and metastasis. Conventional approaches for monitoring oxidative stress in tissue such as high-performance liquid chromatography and immunohistochemistry are bulk measurements and destroy the sample, meaning that longitudinal monitoring of cancer cell heterogeneity remains elusive. Raman spectroscopy has the potential to overcome this challenge, providing a chemically specific, label free readout from single living cells. Here, we applied a standardized protocol for label-free confocal Raman micro-spectroscopy in living cells to monitor oxidative stress in bronchial cells. We used a quartz substrate in a commercial cell chamber contained within a microscope incubator providing culture media for cell maintenance. We studied the effect of a potent reactive oxygen species inducer, tert-butyl hydroperoxide (TBHP), and antioxidant, N-acetyl-L-cysteine (NAC) on living cells from a human bronchial epithelial cells (HBEC). We found that the Raman bands corresponding to nucleic acids, proteins and lipids were significantly different (pmicro-spectroscopy may be able to monitor the biological impact of oxidative and reductive processes in cells, hence enabling longitudinal studies of oxidative stress in therapy resistance and metastasis at the single cell level.

  9. A highly-sensitive label-free biosensor based on two dimensional photonic crystals with negative refraction

    Science.gov (United States)

    Malmir, Narges; Fasihi, Kiazand

    2017-11-01

    In this work, we present a novel high-sensitive optical label-free biosensor based on a two-dimensional photonic crystal (2D PC). The suggested structure is composed of a negative refraction structure in a hexagonal lattice PC, along with a positive refraction structure which is arranged in a square lattice PC. The frequency shift of the transmission peak is measured respect to the changes of refractive indices of the studied materials (the blood plasma, water, dry air and normal air). The studied materials are filled into a W1 line-defect waveguide which is located in the PC structure with positive refraction (the microfluidic nanochannel). Our numerical simulations, which are based on finite-difference time-domain (FDTD) method, show that in the proposed structure, a sensitivity about 1100 nm/RIU and a transmission efficiency more than 75% can be achieved. With this design, to the best of our knowledge, the obtained sensitivity and the transmission efficiency are one of the highest values in the reported PC label-free biosensors.

  10. Quantum Cascade Laser-Based Infrared Microscopy for Label-Free and Automated Cancer Classification in Tissue Sections.

    Science.gov (United States)

    Kuepper, Claus; Kallenbach-Thieltges, Angela; Juette, Hendrik; Tannapfel, Andrea; Großerueschkamp, Frederik; Gerwert, Klaus

    2018-05-16

    A feasibility study using a quantum cascade laser-based infrared microscope for the rapid and label-free classification of colorectal cancer tissues is presented. Infrared imaging is a reliable, robust, automated, and operator-independent tissue classification method that has been used for differential classification of tissue thin sections identifying tumorous regions. However, long acquisition time by the so far used FT-IR-based microscopes hampered the clinical translation of this technique. Here, the used quantum cascade laser-based microscope provides now infrared images for precise tissue classification within few minutes. We analyzed 110 patients with UICC-Stage II and III colorectal cancer, showing 96% sensitivity and 100% specificity of this label-free method as compared to histopathology, the gold standard in routine clinical diagnostics. The main hurdle for the clinical translation of IR-Imaging is overcome now by the short acquisition time for high quality diagnostic images, which is in the same time range as frozen sections by pathologists.

  11. Label-free, multi-contrast optical coherence tomography for study of skin melanoma mice in vivo

    Science.gov (United States)

    Lai, Pei-Yu; Lin, Tim-Han; Chou, Ya-Shuan; Chang, Chung-Hsing; Kuo, Wen-Chuan

    2018-02-01

    The lymphatic system plays an important role in inflammation and cancer such as melanoma. Due to the limitations of current developed imaging techniques, visualization of lymphatic vessels within the tissue in vivo has been challenging. Optical imaging of lymphatic vessel is gaining increased interests because it does not involve any radiation and can achieve very high resolution. Here, we developed a multi-contrast, label-free optical coherence tomography (OCT) imaging technology with an axial resolution of 5 μm and lateral resolution of 7 μm, which is capable of providing microstructural information and microcirculatory system including blood and lymphatic vessels simultaneously. Using this technique, we observed the melanoma mice in vivo. Mice were treated topically on the ear with (Z)-4- Hydroxytamoxifen(4-OHT) to elicit BRAFV600E and to silence Pten expression. Also, to observing the structural information, angiogenesis and lymphangiogenesis in the ear of the induced melanoma mouse can be done. The advantage of using OCT over other imaging modalities is its ability to assess label-free blood flow along with lymphatic vessels simultaneously for imaging the microcirculatory system within tissue beds without any exogenous agents. Because the metastasis of melanoma is highly related to the lymphatic vessels, our findings can be a powerful tool to help the diagnosis of the metastasis melanoma. In the future, this may become a helpful tool for better understanding pathologic mechanisms and treatment technique development in some diseases.

  12. Label-free detection of protein biomolecules secreted from a heart-on-a-chip model for drug cardiotoxicity evaluation

    Science.gov (United States)

    DeLuna, Frank; Zhang, Yu Shrike; Bustamante, Gilbert; Li, Le; Lauderdale, Matthew; Dokmeci, Mehmet R.; Khademhosseini, Ali; Ye, Jing Yong

    2018-02-01

    Efficient methods for the accurate analysis of drug toxicities are in urgent demand as failures of newly discovered drug candidates due to toxic side effects have resulted in about 30% of clinical attrition. The high failure rate is partly due to current inadequate models to study drug side effects, i.e., common animal models may fail due to its misrepresentation of human physiology. Therefore, much effort has been allocated in the development of organ-on-a-chip models which offer a variety of human organ models mimicking a multitude of human physiological conditions. However, it is extremely challenging to analyze the transient and long-term response of the organ models to drug treatments during drug toxicity tests, as the proteins secreted from the organ-on-a-chip model are minute due to its volumetric size, and current methods for detecting said biomolecules are not suitable for real-time monitoring. As protein biomolecules are being continuously secreted from the human organ model, fluorescence techniques are practically impossible to achieve real-time fluorescence labeling in the dynamically changing environment, thus making a label-free approach highly desirable for the organ-on-achip applications. In this paper, we report the use of a photonic-crystal biosensor integrated with a microfluidic system for sensitive label-free bioassays of secreted protein biomolecules from a heart-on-the-chip model created with cardiomyocytes derived from human induced pluripotent stem cells.

  13. Alzheimer's disease evaluation using label-free, stainless, fluorescence to measure tryptophan metabolism along the kynurenine pathway

    Science.gov (United States)

    Sordillo, Laura A.; Zhang, Lin; Shi, Lingyan; Sriramoju, Vidyasagar; Sordillo, Peter P.; Alfano, Robert R.

    2018-02-01

    Under stress conditions, pro-inflammatory cytokines, such as tumor necrosis factor-alpha, interleukin-1 beta, interleukin 6 and interferon gamma are released. It is known that these cytokines stimulate indoleamine 2,3-dioxygenase (IDO) and tryptophan 2,3-dioxygenase (TDO), which increase tryptophan metabolism through the kynurenine pathway, and that this can cause increased production of neurotoxic compounds. Brain tissues from Alzheimer's disease patients and agematched controls were investigated using label-free fluorescence spectroscopy. Tryptophan (exc. 280/ em. 340 nm) and its metabolites (N-formyl-L-kynurenine (exc. 325/em. 434 nm), kynurenine (exc. 365/em. 480 nm) and kynurenic acid (exc. 330/em. 390 nm)) have distinct spectral profiles. Preliminary results show a difference in the optical signatures in three important areas of the brain (hippocampus, BA 9, BA 17) between patients with Alzheimer's disease and agedmatched controls (normal), and a marked relative increase in tryptophan in the Alzheimer's patients. Thus determinations of tryptophan to tryptophan metabolite ratios could potentially be used to measure IDO and TDO activity and the degree of inflammation in the brain. This label-free optical technique may be useful in the study of Alzheimer's and other neurodegenerative diseases.

  14. Label-Free Virus Capture and Release by a Microfluidic Device Integrated with Porous Silicon Nanowire Forest.

    Science.gov (United States)

    Xia, Yiqiu; Tang, Yi; Yu, Xu; Wan, Yuan; Chen, Yizhu; Lu, Huaguang; Zheng, Si-Yang

    2017-02-01

    Viral diseases are perpetual threats to human and animal health. Detection and characterization of viral pathogens require accurate, sensitive, and rapid diagnostic assays. For field and clinical samples, the sample preparation procedures limit the ultimate performance and utility of the overall virus diagnostic protocols. This study presents the development of a microfluidic device embedded with porous silicon nanowire (pSiNW) forest for label-free size-based point-of-care virus capture in a continuous curved flow design. The pSiNW forests with specific interwire spacing are synthesized in situ on both bottom and sidewalls of the microchannels in a batch process. With the enhancement effect of Dean flow, this study demonstrates that about 50% H5N2 avian influenza viruses are physically trapped without device clogging. A unique feature of the device is that captured viruses can be released by inducing self-degradation of the pSiNWs in physiological aqueous environment. About 60% of captured viruses can be released within 24 h for virus culture, subsequent molecular diagnosis, and other virus characterization and analyses. This device performs viable, unbiased, and label-free virus isolation and release. It has great potentials for virus discovery, virus isolation and culture, functional studies of virus pathogenicity, transmission, drug screening, and vaccine development. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Genetic determinants of PAM-dependent DNA targeting and pre-crRNA processing in Sulfolobus islandicus

    DEFF Research Database (Denmark)

    Peng, Wenfang; Li, Huan; Hallstrøm, Søren

    2013-01-01

    -adjacent motif (PAM)-dependent DNA targeting activity and mature CRISPR RNA (crRNA) production in this organism, mutants deleting individual genes of the type IA system or removing each of other Cas modules were constructed. Characterization of these mutants revealed that Cas7, Cas5, Cas6, Cas3' and Cas3......" are essential for PAM-dependent DNA targeting activity, whereas Csa5, along with all other Cas modules, is dispensable for the targeting in the crenarchaeon. Cas6 is implicated as the only enzyme for pre-crRNA processing and the crRNA maturation is independent of the DNA targeting activity. Importantly, we show...

  16. A RNA-DNA Hybrid Aptamer for Nanoparticle-Based Prostate Tumor Targeted Drug Delivery

    Directory of Open Access Journals (Sweden)

    John C. Leach

    2016-03-01

    Full Text Available The side effects of radio- and chemo-therapy pose long-term challenges on a cancer patient’s health. It is, therefore, highly desirable to develop more effective therapies that can specifically target carcinoma cells without damaging normal and healthy cells. Tremendous efforts have been made in the past to develop targeted drug delivery systems for solid cancer treatment. In this study, a new aptamer, A10-3-J1, which recognizes the extracellular domain of the prostate specific membrane antigen (PSMA, was designed. A super paramagnetic iron oxide nanoparticle-aptamer-doxorubicin (SPIO-Apt-Dox was fabricated and employed as a targeted drug delivery platform for cancer therapy. This DNA RNA hybridized aptamer antitumor agent was able to enhance the cytotoxicity of targeted cells while minimizing collateral damage to non-targeted cells. This SPIO-Apt-Dox nanoparticle has specificity to PSMA+ prostate cancer cells. Aptamer inhibited nonspecific uptake of membrane-permeable doxorubic to the non-target cells, leading to reduced untargeted cytotoxicity and endocytic uptake while enhancing targeted cytotoxicity and endocytic uptake. The experimental results indicate that the drug delivery platform can yield statistically significant effectiveness being more cytotoxic to the targeted cells as opposed to the non-targeted cells.

  17. Comparison of a Label-Free Quantitative Proteomic Method Based on Peptide Ion Current Area to the Isotope Coded Affinity Tag Method

    Directory of Open Access Journals (Sweden)

    Young Ah Goo

    2008-01-01

    Full Text Available Recently, several research groups have published methods for the determination of proteomic expression profiling by mass spectrometry without the use of exogenously added stable isotopes or stable isotope dilution theory. These so-called label-free, methods have the advantage of allowing data on each sample to be acquired independently from all other samples to which they can later be compared in silico for the purpose of measuring changes in protein expression between various biological states. We developed label free software based on direct measurement of peptide ion current area (PICA and compared it to two other methods, a simpler label free method known as spectral counting and the isotope coded affinity tag (ICAT method. Data analysis by these methods of a standard mixture containing proteins of known, but varying, concentrations showed that they performed similarly with a mean squared error of 0.09. Additionally, complex bacterial protein mixtures spiked with known concentrations of standard proteins were analyzed using the PICA label-free method. These results indicated that the PICA method detected all levels of standard spiked proteins at the 90% confidence level in this complex biological sample. This finding confirms that label-free methods, based on direct measurement of the area under a single ion current trace, performed as well as the standard ICAT method. Given the fact that the label-free methods provide ease in experimental design well beyond pair-wise comparison, label-free methods such as our PICA method are well suited for proteomic expression profiling of large numbers of samples as is needed in clinical analysis.

  18. Active colloids as mobile microelectrodes for unified label-free selective cargo transport.

    Science.gov (United States)

    Boymelgreen, Alicia M; Balli, Tov; Miloh, Touvia; Yossifon, Gilad

    2018-02-22

    Utilization of active colloids to transport both biological and inorganic cargo has been widely examined in the context of applications ranging from targeted drug delivery to sample analysis. In general, carriers are customized to load one specific target via a mechanism distinct from that driving the transport. Here we unify these tasks and extend loading capabilities to include on-demand selection of multiple nano/micro-sized targets without the need for pre-labelling or surface functionalization. An externally applied electric field is singularly used to drive the active cargo carrier and transform it into a mobile floating electrode that can attract (trap) or repel specific targets from its surface by dielectrophoresis, enabling dynamic control of target selection, loading and rate of transport via the electric field parameters. In the future, dynamic selectivity could be combined with directed motion to develop building blocks for bottom-up fabrication in applications such as additive manufacturing and soft robotics.

  19. Detection of Balamuthia mandrillaris DNA by real-time PCR targeting the RNase P gene

    Directory of Open Access Journals (Sweden)

    Lewin Astrid

    2008-12-01

    Full Text Available Abstract Background The free-living amoeba Balamuthia mandrillaris may cause fatal encephalitis both in immunocompromised and in – apparently – immunocompetent humans and other mammalian species. Rapid, specific, sensitive, and reliable detection requiring little pathogen-specific expertise is an absolute prerequisite for a successful therapy and a welcome tool for both experimental and epidemiological research. Results A real-time polymerase chain reaction assay using TaqMan® probes (real-time PCR was established specifically targeting the RNase P gene of B. mandrillaris amoebae. The assay detected at least 2 (down to 0.5 genomes of B. mandrillaris grown in axenic culture. It did not react with DNA from closely related Acanthamoeba (3 species, nor with DNA from Toxoplasma gondii, Leishmania major, Pneumocystis murina, Mycobacterium bovis (BCG, human brain, various mouse organs, or from human and murine cell lines. The assay efficiently detected B. mandrillaris DNA in spiked cell cultures, spiked murine organ homogenates, B. mandrillaris-infected mice, and CNS tissue-DNA preparations from 2 patients with proven cerebral balamuthiasis. This novel primer set was successfully combined with a published set that targets the B. mandrillaris 18S rRNA gene in a duplex real-time PCR assay to ensure maximum specificity and as a precaution against false negative results. Conclusion A real-time PCR assay for B. mandrillaris amoebae is presented, that is highly specific, sensitive, and reliable and thus suited both for diagnosis and for research.

  20. Prioritizing multiple therapeutic targets in parallel using automated DNA-encoded library screening

    Science.gov (United States)

    Machutta, Carl A.; Kollmann, Christopher S.; Lind, Kenneth E.; Bai, Xiaopeng; Chan, Pan F.; Huang, Jianzhong; Ballell, Lluis; Belyanskaya, Svetlana; Besra, Gurdyal S.; Barros-Aguirre, David; Bates, Robert H.; Centrella, Paolo A.; Chang, Sandy S.; Chai, Jing; Choudhry, Anthony E.; Coffin, Aaron; Davie, Christopher P.; Deng, Hongfeng; Deng, Jianghe; Ding, Yun; Dodson, Jason W.; Fosbenner, David T.; Gao, Enoch N.; Graham, Taylor L.; Graybill, Todd L.; Ingraham, Karen; Johnson, Walter P.; King, Bryan W.; Kwiatkowski, Christopher R.; Lelièvre, Joël; Li, Yue; Liu, Xiaorong; Lu, Quinn; Lehr, Ruth; Mendoza-Losana, Alfonso; Martin, John; McCloskey, Lynn; McCormick, Patti; O'Keefe, Heather P.; O'Keeffe, Thomas; Pao, Christina; Phelps, Christopher B.; Qi, Hongwei; Rafferty, Keith; Scavello, Genaro S.; Steiginga, Matt S.; Sundersingh, Flora S.; Sweitzer, Sharon M.; Szewczuk, Lawrence M.; Taylor, Amy; Toh, May Fern; Wang, Juan; Wang, Minghui; Wilkins, Devan J.; Xia, Bing; Yao, Gang; Zhang, Jean; Zhou, Jingye; Donahue, Christine P.; Messer, Jeffrey A.; Holmes, David; Arico-Muendel, Christopher C.; Pope, Andrew J.; Gross, Jeffrey W.; Evindar, Ghotas

    2017-07-01

    The identification and prioritization of chemically tractable therapeutic targets is a significant challenge in the discovery of new medicines. We have developed a novel method that rapidly screens multiple proteins in parallel using DNA-encoded library technology (ELT). Initial efforts were focused on the efficient discovery of antibacterial leads against 119 targets from Acinetobacter baumannii and Staphylococcus aureus. The success of this effort led to the hypothesis that the relative number of ELT binders alone could be used to assess the ligandability of large sets of proteins. This concept was further explored by screening 42 targets from Mycobacterium tuberculosis. Active chemical series for six targets from our initial effort as well as three chemotypes for DHFR from M. tuberculosis are reported. The findings demonstrate that parallel ELT selections can be used to assess ligandability and highlight opportunities for successful lead and tool discovery.

  1. Assembling the Streptococcus thermophilus clustered regularly interspaced short palindromic repeats (CRISPR) array for multiplex DNA targeting.

    Science.gov (United States)

    Guo, Lijun; Xu, Kun; Liu, Zhiyuan; Zhang, Cunfang; Xin, Ying; Zhang, Zhiying

    2015-06-01

    In addition to the advantages of scalable, affordable, and easy to engineer, the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) technology is superior for multiplex targeting, which is laborious and inconvenient when achieved by cloning multiple gRNA expressing cassettes. Here, we report a simple CRISPR array assembling method which will facilitate multiplex targeting usage. First, the Streptococcus thermophilus CRISPR3/Cas locus was cloned. Second, different CRISPR arrays were assembled with different crRNA spacers. Transformation assays using different Escherichia coli strains demonstrated efficient plasmid DNA targeting, and we achieved targeting efficiency up to 95% with an assembled CRISPR array with three crRNA spacers. Copyright © 2015 Elsevier Inc. All rights reserved.

  2. Dendritic Cell Targeted Chitosan Nanoparticles for Nasal DNA Immunization against SARS CoV Nucleocapsid Protein

    OpenAIRE

    Raghuwanshi, Dharmendra; Mishra, Vivek; Das, Dipankar; Kaur, Kamaljit; Suresh, Mavanur R.

    2012-01-01

    This work investigates the formulation and in vivo efficacy of dendritic cell (DC) targeted plasmid DNA loaded biotinylated chitosan nanoparticles for nasal immunization against nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus (SARS-CoV) as antigen. The induction of antigen-specific mucosal and systemic immune response at the site of virus entry is a major challenge for vaccine design. Here, we designed a strategy for non-invasive receptor mediated gene delivery to na...

  3. Dynamic conformational change regulates the protein-DNA recognition: an investigation on binding of a Y-family polymerase to its target DNA.

    Directory of Open Access Journals (Sweden)

    Xiakun Chu

    2014-09-01

    Full Text Available Protein-DNA recognition is a central biological process that governs the life of cells. A protein will often undergo a conformational transition to form the functional complex with its target DNA. The protein conformational dynamics are expected to contribute to the stability and specificity of DNA recognition and therefore may control the functional activity of the protein-DNA complex. Understanding how the conformational dynamics influences the protein-DNA recognition is still challenging. Here, we developed a two-basin structure-based model to explore functional dynamics in Sulfolobus solfataricus DNA Y-family polymerase IV (DPO4 during its binding to DNA. With explicit consideration of non-specific and specific interactions between DPO4 and DNA, we found that DPO4-DNA recognition is comprised of first 3D diffusion, then a short-range adjustment sliding on DNA and finally specific binding. Interestingly, we found that DPO4 is under a conformational equilibrium between multiple states during the binding process and the distributions of the conformations vary at different binding stages. By modulating the strength of the electrostatic interactions, the flexibility of the linker, and the conformational dynamics in DPO4, we drew a clear picture on how DPO4 dynamically regulates the DNA recognition. We argue that the unique features of flexibility and conformational dynamics in DPO4-DNA recognition have direct implications for low-fidelity translesion DNA synthesis, most of which is found to be accomplished by the Y-family DNA polymerases. Our results help complete the description of the DNA synthesis process for the Y-family polymerases. Furthermore, the methods developed here can be widely applied for future investigations on how various proteins recognize and bind specific DNA substrates.

  4. Promotion of BRCA2-Dependent Homologous Recombination by DSS1 via RPA Targeting and DNA Mimicry.

    Science.gov (United States)

    Zhao, Weixing; Vaithiyalingam, Sivaraja; San Filippo, Joseph; Maranon, David G; Jimenez-Sainz, Judit; Fontenay, Gerald V; Kwon, Youngho; Leung, Stanley G; Lu, Lucy; Jensen, Ryan B; Chazin, Walter J; Wiese, Claudia; Sung, Patrick

    2015-07-16

    The tumor suppressor BRCA2 is thought to facilitate the handoff of ssDNA from replication protein A (RPA) to the RAD51 recombinase during DNA break and replication fork repair by homologous recombination. However, we find that RPA-RAD51 exchange requires the BRCA2 partner DSS1. Biochemical, structural, and in vivo analyses reveal that DSS1 allows the BRCA2-DSS1 complex to physically and functionally interact with RPA. Mechanistically, DSS1 acts as a DNA mimic to attenuate the affinity of RPA for ssDNA. A mutation in the solvent-exposed acidic domain of DSS1 compromises the efficacy of RPA-RAD51 exchange. Thus, by targeting RPA and mimicking DNA, DSS1 functions with BRCA2 in a two-component homologous recombination mediator complex in genome maintenance and tumor suppression. Our findings may provide a paradigm for understanding the roles of DSS1 in other biological processes. Copyright © 2015 Elsevier Inc. All rights reserved.

  5. Targeted DNA vaccines for enhanced induction of idiotype-specific B and T cells

    International Nuclear Information System (INIS)

    Fredriksen, Agnete B.; Sandlie, Inger; Bogen, Bjarne

    2012-01-01

    Background: Idiotypes (Id) are antigenic determinants localized in variable (V) regions of Ig. Id-specific T and B cells (antibodies) play a role in immunotherapy of Id + tumors. However, vaccine strategies that enhance Id-specific responses are needed. Methods: Id + single-chain fragment variable (scFv) from multiple myelomas and B cell lymphomas were prepared in a fusion format that bivalently target surface molecules on antigen-presenting cells (APC). APC-specific targeting units were either scFv from APC-specific mAb (anti-MHC II, anti-CD40) or chemokines (MIP-1α, RANTES). Homodimeric Id-vaccines were injected intramuscularly or intradermally as plasmids in mice, combined with electroporation. Results: (i) Transfected cells secreted plasmid-encoded Id + fusion proteins to extracellular fluid followed by binding of vaccine molecules to APC. (ii) Targeted vaccine molecules increased Id-specific B and T cell responses. (iii) Bivalency and xenogeneic sequences both contributed to enhanced responses. (iv) Targeted Id DNA vaccines induced tumor resistance against challenges with Id + tumors. (v) Human MIP-1α targeting units enhanced Id-specific responses in mice, due to a cross reaction with murine chemokine receptors. Thus, targeted vaccines designed for humans can be quality tested in mice. (vi) Human Id + scFv from four multiple myeloma patients were inserted into the vaccine format and were successfully tested in mice. (vii) Human MIP-1α vaccine proteins enhanced human T cell responses in vitro. (viii) A hypothetical model for how the APC-targeted vaccine molecules enhance Id-specific T and B cells is presented. Conclusion: Targeted DNA Id-vaccines show promising results in preclinical studies, paving the way for testing in patients.

  6. Targeted DNA vaccines for enhanced induction of idiotype-specific B and T cells

    Energy Technology Data Exchange (ETDEWEB)

    Fredriksen, Agnete B.; Sandlie, Inger; Bogen, Bjarne, E-mail: bjarne.bogen@medisin.uio.no [Centre for Immune Regulation, Institute of Immunology, University of Oslo and Oslo University Hospital, Oslo (Norway)

    2012-10-30

    Background: Idiotypes (Id) are antigenic determinants localized in variable (V) regions of Ig. Id-specific T and B cells (antibodies) play a role in immunotherapy of Id{sup +} tumors. However, vaccine strategies that enhance Id-specific responses are needed. Methods: Id{sup +} single-chain fragment variable (scFv) from multiple myelomas and B cell lymphomas were prepared in a fusion format that bivalently target surface molecules on antigen-presenting cells (APC). APC-specific targeting units were either scFv from APC-specific mAb (anti-MHC II, anti-CD40) or chemokines (MIP-1α, RANTES). Homodimeric Id-vaccines were injected intramuscularly or intradermally as plasmids in mice, combined with electroporation. Results: (i) Transfected cells secreted plasmid-encoded Id{sup +} fusion proteins to extracellular fluid followed by binding of vaccine molecules to APC. (ii) Targeted vaccine molecules increased Id-specific B and T cell responses. (iii) Bivalency and xenogeneic sequences both contributed to enhanced responses. (iv) Targeted Id DNA vaccines induced tumor resistance against challenges with Id{sup +} tumors. (v) Human MIP-1α targeting units enhanced Id-specific responses in mice, due to a cross reaction with murine chemokine receptors. Thus, targeted vaccines designed for humans can be quality tested in mice. (vi) Human Id{sup +} scFv from four multiple myeloma patients were inserted into the vaccine format and were successfully tested in mice. (vii) Human MIP-1α vaccine proteins enhanced human T cell responses in vitro. (viii) A hypothetical model for how the APC-targeted vaccine molecules enhance Id-specific T and B cells is presented. Conclusion: Targeted DNA Id-vaccines show promising results in preclinical studies, paving the way for testing in patients.

  7. Label-free and enzyme-free detection of transcription factors with graphene oxide fluorescence switch-based multifunctional G-quadruplex-hairpin probe.

    Science.gov (United States)

    Zhu, Desong; Wang, Lei; Xu, Xiaowen; Jiang, Wei

    2016-01-15

    Transcription factors (TFs) play pivotal roles in the regulation of a variety of essential cellular processes and some of them have been recognized as potential diagnostic markers and therapeutic targets of some diseases. Sensitive and accurate detection of TFs is of great importance to better understanding their roles in gene regulation and evaluation of disease state. Here, we developed a simple, label-free and enzyme-free new fluorescent strategy for the detection of TFs by graphene oxide (GO) fluorescence switch-based multifunctional G-quadruplex-hairpin probe (MGHP). The MGHP possessed of three functions simultaneously, adsorbing onto GO with the loop part, binding to target with the stem part and serving as signal carrier with the terminal G-quadruplex. First, the MGHP was adsorbed quickly to GO. Next, the TF bound to the stem part of MGHP to form a huge target-MGHP complex, which led to desorption of the complex from GO. Finally, NMM was inserted into G-quadruplex in the complex to yield an enhanced fluorescence response. The GO used here, as a fluorescence switch, could quickly and efficiently quench the fluorescence of NMM inserted into the MGHP absorbed on the GO, guaranteeing a high signal-to-noise ratio. Sensitive detection of purified NF-κB p50 and HeLa cell nuclear extracts were achieved with detection limits of 0.2nM and 7.8ng/µL, respectively. Moreover, this proposed strategy could be used to screen inhibitors of NF-κB p50 activity. The strategy proposed here might offer a new potential approach for reliable quantification of TFs in clinical diagnostics and treatment research of some diseases. Copyright © 2015 Elsevier B.V. All rights reserved.

  8. Inspecting Targeted Deep Sequencing of Whole Genome Amplified DNA Versus Fresh DNA for Somatic Mutation Detection: A Genetic Study in Myelodysplastic Syndrome Patients.

    Science.gov (United States)

    Palomo, Laura; Fuster-Tormo, Francisco; Alvira, Daniel; Ademà, Vera; Armengol, María Pilar; Gómez-Marzo, Paula; de Haro, Nuri; Mallo, Mar; Xicoy, Blanca; Zamora, Lurdes; Solé, Francesc

    2017-08-01

    Whole genome amplification (WGA) has become an invaluable method for preserving limited samples of precious stock material and has been used during the past years as an alternative tool to increase the amount of DNA before library preparation for next-generation sequencing. Myelodysplastic syndromes (MDS) are a group of clonal hematopoietic stem cell disorders characterized by presenting somatic mutations in several myeloid-related genes. In this work, targeted deep sequencing has been performed on four paired fresh DNA and WGA DNA samples from bone marrow of MDS patients, to assess the feasibility of using WGA DNA for detecting somatic mutations. The results of this study highlighted that, in general, the sequencing and alignment statistics of fresh DNA and WGA DNA samples were similar. However, after variant calling and when considering variants detected at all frequencies, there was a high level of discordance between fresh DNA and WGA DNA (overall, a higher number of variants was detected in WGA DNA). After proper filtering, a total of three somatic mutations were detected in the cohort. All somatic mutations detected in fresh DNA were also identified in WGA DNA and validated by whole exome sequencing.

  9. Targeting GLI by GANT61 involves mechanisms dependent on inhibition of both transcription and DNA licensing.

    Science.gov (United States)

    Zhang, Ruowen; Wu, Jiahui; Ferrandon, Sylvain; Glowacki, Katie J; Houghton, Janet A

    2016-12-06

    The GLI genes are transcription factors and in cancers are oncogenes, aberrantly and constitutively activated. GANT61, a specific GLI inhibitor, has induced extensive cytotoxicity in human models of colon cancer. The FOXM1 promoter was determined to be a transcriptional target of GLI1. In HT29 cells, inhibition of GLI1 binding at the GLI consensus sequence by GANT61 led to inhibited binding of Pol II, the pause-release factors DSIF, NELF and p-TEFb. The formation of R-loops (RNA:DNA hybrids, ssDNA), were reduced by GANT61 at the FOXM1 promoter. Pretreatment of HT29 cells with α-amanitin reduced GANT61-induced γH2AX foci. Co-localization of GLI1 and BrdU foci, inhibited by GANT61, indicated GLI1 and DNA replication to be linked. By co-immunoprecipitation and confocal microscopy, GLI1 co-localized with the DNA licensing factors ORC4, CDT1, and MCM2. Significant co-localization of GLI1 and ORC4 was inhibited by GANT61, and enrichment of ORC4 occurred at the GLI binding site in the FOXM1 promoter. CDT1 was found to be a transcription target of GLI1. Overexpression of CDT1 in HT29 and SW480 cells reduced GANT61-induced cell death, gH2AX foci, and cleavage of caspase-3. Data demonstrate involvement of transcription and of DNA replication licensing factors by non-transcriptional and transcriptional mechanisms in the GLI-dependent mechanism of action of GANT61.

  10. Label free luminescence strategy for sensitive detection of ATP using aptamer-Ru(II) complexes

    Energy Technology Data Exchange (ETDEWEB)

    Babu, Eththilu [Department of Physical Che mistry, School of Chemistry, Madurai Kamaraj University, Madurai 625021, Tamil Nadu (India); Muthu Mareeswaran, Paulpandian [Department of Physical Che mistry, School of Chemistry, Madurai Kamaraj University, Madurai 625021, Tamil Nadu (India); Department of Industrial Chemistry, Alagappa Univesity, Karaikudi 630003, Tamil Nadu (India); Ramdass, Arumugam [Department of Physical Che mistry, School of Chemistry, Madurai Kamaraj University, Madurai 625021, Tamil Nadu (India); Research Department of Chemistry, Aditanar College of Arts and Science, Tiruchendur 628216, Tamil Nadu (India); Ramesh, Pandian [UCIBIO-REQUIMTE, Departmento de Química, Faculdade de Ciências e Tecnologia, Universidade NOVA de Lisboa, 2829-516 Caparica (Portugal); Rajagopal, Seenivasan, E-mail: rajagopalseenivasan@yahoo.com [Department of Physical Che mistry, School of Chemistry, Madurai Kamaraj University, Madurai 625021, Tamil Nadu (India)

    2016-07-15

    A simple and sensitive aptamer-based luminescence strategy for ATP detection is developed using Ru(II) complexes as probe molecule. It is based on the fact that Ru(II)-dppz complexes show the light switching behavior with DNA aptamers and found to show significant luminescence spectral change on the addition of ATP molecules. The binding efficiencies of aptamer with ATP, ADP and AMP are calculated and compared. The structural change of aptamer is also studied using circular dichroism (CD) spectral techniques. Moreover, the binding nature of aptamer with ATP, ADP and AMP is demonstrated by computational techniques. The proposed strategy was successfully applied to the detection of ATP.

  11. Label free luminescence strategy for sensitive detection of ATP using aptamer-Ru(II) complexes

    International Nuclear Information System (INIS)

    Babu, Eththilu; Muthu Mareeswaran, Paulpandian; Ramdass, Arumugam; Ramesh, Pandian; Rajagopal, Seenivasan

    2016-01-01

    A simple and sensitive aptamer-based luminescence strategy for ATP detection is developed using Ru(II) complexes as probe molecule. It is based on the fact that Ru(II)-dppz complexes show the light switching behavior with DNA aptamers and found to show significant luminescence spectral change on the addition of ATP molecules. The binding efficiencies of aptamer with ATP, ADP and AMP are calculated and compared. The structural change of aptamer is also studied using circular dichroism (CD) spectral techniques. Moreover, the binding nature of aptamer with ATP, ADP and AMP is demonstrated by computational techniques. The proposed strategy was successfully applied to the detection of ATP.

  12. Targeting Extracellular DNA to Deliver IGF-1 to the Injured Heart

    Science.gov (United States)

    Khan, Raffay S.; Martinez, Mario D.; Sy, Jay C.; Pendergrass, Karl D.; Che, Pao-Lin; Brown, Milton E.; Cabigas, E. Bernadette; Dasari, Madhuri; Murthy, Niren; Davis, Michael E.

    2014-03-01

    There is a great need for the development of therapeutic strategies that can target biomolecules to damaged myocardium. Necrosis of myocardium during a myocardial infarction (MI) is characterized by extracellular release of DNA, which can serve as a potential target for ischemic tissue. Hoechst, a histological stain that binds to double-stranded DNA can be conjugated to a variety of molecules. Insulin-like growth factor-1 (IGF-1), a small protein/polypeptide with a short circulating-half life is cardioprotective following MI but its clinical use is limited by poor delivery, as intra-myocardial injections have poor retention and chronic systemic presence has adverse side effects. Here, we present a novel delivery vehicle for IGF-1, via its conjugation to Hoechst for targeting infarcted tissue. Using a mouse model of ischemia-reperfusion, we demonstrate that intravenous delivery of Hoechst-IGF-1 results in activation of Akt, a downstream target of IGF-1 and protects from cardiac fibrosis and dysfunction following MI.

  13. Nonviral Gene Targeting at rDNA Locus of Human Mesenchymal Stem Cells

    Directory of Open Access Journals (Sweden)

    Youjin Hu

    2013-01-01

    Full Text Available Background. Genetic modification, such as the addition of exogenous genes to the MSC genome, is crucial to their use as cellular vehicles. Due to the risks associated with viral vectors such as insertional mutagenesis, the safer nonviral vectors have drawn a great deal of attention. Methods. VEGF, bFGF, vitamin C, and insulin-transferrin-selenium-X were supplemented in the MSC culture medium. The cells’ proliferation and survival capacity was measured by MTT, determination of the cumulative number of cells, and a colony-forming efficiency assay. The plasmid pHr2-NL was constructed and nucleofected into MSCs. The recombinants were selected using G418 and characterized using PCR and Southern blotting. Results. BFGF is critical to MSC growth and it acted synergistically with vitamin C, VEGF, and ITS-X, causing the cells to expand significantly. The neomycin gene was targeted to the rDNA locus of human MSCs using a nonviral human ribosomal targeting vector. The recombinant MSCs retained multipotential differentiation capacity, typical levels of hMSC surface marker expression, and a normal karyotype, and none were tumorigenic in nude mice. Conclusions. Exogenous genes can be targeted to the rDNA locus of human MSCs while maintaining the characteristics of MSCs. This is the first nonviral gene targeting of hMSCs.

  14. Identification and characterization of DNAzymes targeting DNA methyltransferase I for suppressing bladder cancer proliferation

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Xiangbo; Zhang, Lu; Ding, Nianhua; Yang, Xinghui; Zhang, Jin; He, Jiang; Li, Zhi; Sun, Lun-Quan, E-mail: lunquansun@csu.edu.cn

    2015-05-29

    Epigenetic inactivation of genes plays a critical role in many important human diseases, especially in cancer. A core mechanism for epigenetic inactivation of the genes is methylation of CpG islands in genome DNA, which is catalyzed by DNA methyltransferases (DNMTs). The inhibition of DNMTs may lead to demethylation and expression of the silenced tumor suppressor genes. Although DNMT inhibitors are currently being developed as potential anticancer agents, only limited success is achieved due to substantial toxicity. Here, we utilized a multiplex selection system to generate efficient RNA-cleaving DNAzymes targeting DNMT1. The lead molecule from the selection was shown to possess efficient kinetic profiles and high efficiency in inhibiting the enzyme activity. Transfection of the DNAzyme caused significant down-regulation of DNMT1 expression and reactivation of p16 gene, resulting in reduced cell proliferation of bladder cancers. This study provides an alternative for targeting DNMTs for potential cancer therapy. - Highlights: • Identified DNMT1-targeted DNAzymes by multiplex selection system. • Biochemically characterized a lead DNAzyme with high kinetic efficiency. • Validated DNMT1-targeted DNAzyme in its enzymatic and cellular activities.

  15. Detection and size analysis of proteins with switchable DNA layers.

    Science.gov (United States)

    Rant, Ulrich; Pringsheim, Erika; Kaiser, Wolfgang; Arinaga, Kenji; Knezevic, Jelena; Tornow, Marc; Fujita, Shozo; Yokoyama, Naoki; Abstreiter, Gerhard

    2009-04-01

    We introduce a chip-compatible scheme for the label-free detection of proteins in real-time that is based on the electrically driven conformation switching of DNA oligonucleotides on metal surfaces. The switching behavior is a sensitive indicator for the specific recognition of IgG antibodies and antibody fragments, which can be detected in quantities of less than 10(-18) mol on the sensor surface. Moreover, we show how the dynamics of the induced molecular motion can be monitored by measuring the high-frequency switching response. When proteins bind to the layer, the increase in hydrodynamic drag slows the switching dynamics, which allows us to determine the size of the captured proteins. We demonstrate the identification of different antibody fragments by means of their kinetic fingerprint. The switchDNA method represents a generic approach to simultaneously detect and size target molecules using a single analytical platform.

  16. Functionalized gold nanostars for label-free detection of PKA phosphorylation using surface-enhanced Raman spectroscopy

    Science.gov (United States)

    He, Shuai; Kah, James C. Y.

    2017-04-01

    Protein phosphorylation controls fundamental biological processes. Dysregulation of protein kinase is associated with a series of human diseases including cancer. Protein kinase A (PKA) activity has been reported to serve as a potential prognostic marker for cancer. To this end, we developed a non-radioactive, rapid, cheap and robust scheme based on surface-enhanced Raman spectroscopy (SERS) for label-free detection of PKA phosphorylation using gold nanostars (AuNS) functionalized with BSA-kemptide. While bovine serum albumin (BSA) proteins stabilized the AuNS, kemptide, which is a high affinity substrate peptide specific for PKA, were phosphorylated in vitro to generate Raman signals that were identified by performing principal component analysis (PCA) on the acquired SERS spectra.

  17. Assessing Collagen and Elastin Pressure-Dependent Microarchitectures in Live, Human Resistance Arteries by Label-Free Fluorescence Microscopy

    DEFF Research Database (Denmark)

    Bloksgaard, Maria; Thorsted, Bjarne; Brewer, Jonathan R.

    2017-01-01

    The pathogenic contribution of resistance artery remodeling is documented in essential hypertension, diabetes and the metabolic syndrome. Investigations and development of microstructurally motivated mathematical models for understanding the mechanical properties of human resistance arteries...... in health and disease have the potential to aid understanding how disease and medical treatments affect the human microcirculation. To develop these mathematical models, it is essential to decipher the relationship between the mechanical and microarchitectural properties of the microvascular wall....... In this work, we describe an ex vivo method for passive mechanical testing and simultaneous label-free three-dimensional imaging of the microarchitecture of elastin and collagen in the arterial wall of isolated human resistance arteries. The imaging protocol can be applied to resistance arteries of any species...

  18. Label-free optical imaging technologies for rapid translation and use during intraoperative surgical and tumor margin assessment

    Science.gov (United States)

    Boppart, Stephen A.; Brown, J. Quincy; Farah, Camile S.; Kho, Esther; Marcu, Laura; Saunders, Christobel M.; Sterenborg, Henricus J. C. M.

    2018-02-01

    The biannual International Conference on Biophotonics was recently held on April 30 to May 1, 2017, in Fremantle, Western Australia. This continuing conference series brought together key opinion leaders in biophotonics to present their latest results and, importantly, to participate in discussions on the future of the field and what opportunities exist when we collectively work together for using biophotonics for biological discovery and medical applications. One session in this conference, entitled "Tumor Margin Identification: Critiquing Technologies," challenged invited speakers and attendees to review and critique representative label-free optical imaging technologies and their application for intraoperative assessment and guidance in surgical oncology. We are pleased to share a summary in this outlook paper, with the intent to motivate more research inquiry and investigations, to challenge these and other optical imaging modalities to evaluate and improve performance, to spur translation and adoption, and ultimately, to improve the care and outcomes of patients.

  19. A Label-Free and Ultrasensitive Immunosensor for Detection of Human Chorionic Gonadotrophin Based on Graphene FETs.

    Science.gov (United States)

    Islam, Kamrul; Suhail, Ahmed; Pan, Genhua

    2017-07-12

    We report on a label-free immunosensor based on graphene field effect transistors (G-FETs) for the ultrasensitive detection of Human Chorionic Gonadotrophin (hCG), as an indicator of pregnancy and related disorders, such as actopic pregnancy, choriocarcinoma and orchic teratoma. Pyrene based bioactive ester was non-covalently anchored onto the graphene channel in order to retain the sp² lattice. The G-FET transfer characteristics showed repeatable and reliable responses in all surface modifying steps using a direct current (DC) readout system. The hCG concentration gradient showed a detection limit of ~1 pg·mL -1 . The proposed method facilitates the cost-effective and viable production of graphene point-of-care devices for clinical diagnosis.

  20. In vivo, label-free, three-dimensional quantitative imaging of liver surface using multi-photon microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Zhuo, Shuangmu, E-mail: shuangmuzhuo@gmail.com, E-mail: hanry-yu@nuhs.edu.sg [Biosystems and Micromechanics IRG, Singapore-MIT Alliance for Research and Technology, 1 CREATE Way, #04-13/14 Enterprise Wing, 138602 Singapore (Singapore); Institute of Laser and Optoelectronics Technology, Fujian Normal University, Fuzhou 350007 (China); Yan, Jie [Biosystems and Micromechanics IRG, Singapore-MIT Alliance for Research and Technology, 1 CREATE Way, #04-13/14 Enterprise Wing, 138602 Singapore (Singapore); Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, #04-01, 138669 Singapore (Singapore); Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, 14 Medical Drive, MD 11 #04-01A, 117599 Singapore (Singapore); Kang, Yuzhan [Biosystems and Micromechanics IRG, Singapore-MIT Alliance for Research and Technology, 1 CREATE Way, #04-13/14 Enterprise Wing, 138602 Singapore (Singapore); Xu, Shuoyu [Biosystems and Micromechanics IRG, Singapore-MIT Alliance for Research and Technology, 1 CREATE Way, #04-13/14 Enterprise Wing, 138602 Singapore (Singapore); Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, #04-01, 138669 Singapore (Singapore); Computation and System Biology Program, Singapore-MIT Alliance, 4 Engineering Drive 3, E4-04-10, 117576 Singapore (Singapore); Peng, Qiwen [Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, #04-01, 138669 Singapore (Singapore); Computation and System Biology Program, Singapore-MIT Alliance, 4 Engineering Drive 3, E4-04-10, 117576 Singapore (Singapore); Mechanobiology Institute, 5A Engineering Drive 1, T-Lab #05-01, 117411 Singapore (Singapore); and others

    2014-07-14

    Various structural features on the liver surface reflect functional changes in the liver. The visualization of these surface features with molecular specificity is of particular relevance to understanding the physiology and diseases of the liver. Using multi-photon microscopy (MPM), we have developed a label-free, three-dimensional quantitative and sensitive method to visualize various structural features of liver surface in living rat. MPM could quantitatively image the microstructural features of liver surface with respect to the sinuosity of collagen fiber, the elastic fiber structure, the ratio between elastin and collagen, collagen content, and the metabolic state of the hepatocytes that are correlative with the pathophysiologically induced changes in the regions of interest. This study highlights the potential of this technique as a useful tool for pathophysiological studies and possible diagnosis of the liver diseases with further development.

  1. A Label-Free and Ultrasensitive Immunosensor for Detection of Human Chorionic Gonadotrophin Based on Graphene FETs

    Directory of Open Access Journals (Sweden)

    Kamrul Islam

    2017-07-01

    Full Text Available We report on a label-free immunosensor based on graphene field effect transistors (G-FETs for the ultrasensitive detection of Human Chorionic Gonadotrophin (hCG, as an indicator of pregnancy and related disorders, such as actopic pregnancy, choriocarcinoma and orchic teratoma. Pyrene based bioactive ester was non-covalently anchored onto the graphene channel in order to retain the sp2 lattice. The G-FET transfer characteristics showed repeatable and reliable responses in all surface modifying steps using a direct current (DC readout system. The hCG concentration gradient showed a detection limit of ~1 pg·mL−1. The proposed method facilitates the cost-effective and viable production of graphene point-of-care devices for clinical diagnosis.

  2. Nanomechanical microcantilever operated in vibration modes with use of RNA aptamer as receptor molecules for label-free detection of HCV helicase.

    Science.gov (United States)

    Hwang, Kyo Seon; Lee, Sang-Myung; Eom, Kilho; Lee, Jeong Hoon; Lee, Yoon-Sik; Park, Jung Ho; Yoon, Dae Sung; Kim, Tae Song

    2007-11-30

    We report the nanomechanical microcantilevers operated in vibration modes (oscillation) with use of RNA aptamers as receptor molecules for label-free detection of hepatitis C virus (HCV) helicase. The nanomechanical detection principle is that the ligand-receptor binding on the microcantilever surface induces the dynamic response change of microcantilevers. We implemented the label-free detection of HCV helicase in the low concentration as much as 100 pg/ml from measuring the dynamic response change of microcantilevers. Moreover, from the recent studies showing that the ligand-receptor binding generates the surface stress on the microcantilever, we estimate the surface stress, on the oscillating microcantilevers, induced by ligand-receptor binding, i.e. binding between HCV helicase and RNA aptamer. In this article, it is suggested that the oscillating microcantilevers with use of RNA aptamers as receptor molecules may enable one to implement the sensitive label-free detection of very small amount of small-scale proteins.

  3. PML-RARA-targeted DNA vaccine induces protective immunity in a mouse model of leukemia.

    Science.gov (United States)

    Padua, Rose Ann; Larghero, Jerome; Robin, Marie; le Pogam, Carol; Schlageter, Marie-Helene; Muszlak, Sacha; Fric, Jan; West, Robert; Rousselot, Philippe; Phan, Thi Hai; Mudde, Liesbeth; Teisserenc, Helene; Carpentier, Antoine F; Kogan, Scott; Degos, Laurent; Pla, Marika; Bishop, J Michael; Stevenson, Freda; Charron, Dominique; Chomienne, Christine

    2003-11-01

    Despite improved molecular characterization of malignancies and development of targeted therapies, acute leukemia is not curable and few patients survive more than 10 years after diagnosis. Recently, combinations of different therapeutic strategies (based on mechanisms of apoptosis, differentiation and cytotoxicity) have significantly increased survival. To further improve outcome, we studied the potential efficacy of boosting the patient's immune response using specific immunotherapy. In an animal model of acute promyelocytic leukemia, we developed a DNA-based vaccine by fusing the human promyelocytic leukemia-retinoic acid receptor-alpha (PML-RARA) oncogene to tetanus fragment C (FrC) sequences. We show for the first time that a DNA vaccine specifically targeted to an oncoprotein can have a pronounced effect on survival, both alone and when combined with all-trans retinoic acid (ATRA). The survival advantage is concomitant with time-dependent antibody production and an increase in interferon-gamma (IFN-gamma). We also show that ATRA therapy on its own triggers an immune response in this model. When DNA vaccination and conventional ATRA therapy are combined, they induce protective immune responses against leukemia progression in mice and may provide a new approach to improve clinical outcome in human leukemia.

  4. Chitosan-based DNA delivery vector targeted to gonadotropin-releasing hormone (GnRH) receptor.

    Science.gov (United States)

    Boonthum, Chatwalee; Namdee, Katawut; Boonrungsiman, Suwimon; Chatdarong, Kaywalee; Saengkrit, Nattika; Sajomsang, Warayuth; Ponglowhapan, Suppawiwat; Yata, Teerapong

    2017-02-10

    The main purpose of this study was to investigate the application of modified chitosan as a potential vector for gene delivery to gonadotropin-releasing hormone receptor (GnRHR)-expressing cells. Such design of gene carrier could be useful in particular for gene therapy for cancers related to the reproductive system, gene disorders of sexual development, and contraception and fertility control. In this study, a decapeptide GnRH was successfully conjugated to chitosan (CS) as confirmed by proton nuclear magnetic resonance spectroscopy ( 1 H NMR) and Attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR). The synthesized GnRH-conjugated chitosan (GnRH-CS) was able to condense DNA to form positively charged nanoparticles and specifically deliver plasmid DNA to targeted cells in both two-dimensional (2D) and three-dimensional (3D) cell cultures systems. Importantly, GnRH-CS exhibited higher transfection activity compared to unmodified CS. In conclusion, GnRH-conjugated chitosan can be a promising carrier for targeted DNA delivery to GnRHR-expressing cells. Copyright © 2016 Elsevier Ltd. All rights reserved.

  5. Expression profiling on high-density DNA grids to detect novel targets in dendritic cells

    International Nuclear Information System (INIS)

    Weissmann, M.

    2000-10-01

    Gene expression analyzes on a large scale using DNA microarrays is a novel approach to study transcription of thousands of genes in parallel. By comparing gene expression profiles of different cell-types and of cells in different activation, novel regulatory networks will be identified that are unique to a cell-type and hence, important in its biological function. Among the differentially expressed genes many novel drug targets will be found. The Genetic department of the Novartis Research Institute was following this approach to identify novel genes, which are critical in the antigen presenting function of DCs and could become promising drug targets. Drugs that modulate effector functions of DCs towards induction of energy or tolerance in T-cells could be useful in the treatment of chronic inflammatory or autoimmune diseases. By using specific robotics equipment high-density cDNA grids on nylon membranes have been produced for hybridizations with various radioactive labeled DNA probes. By our format, based on 384 well plates and limited by the resolution power of our current image analysis software, 27.648 cDNA clones, bacterial colonies or pure DNA, were spotted on one filter. For RNA profiling, we generated filters containing a collection of genes expressed in peripheral blood DCs or monocytes and characterized by oligonucleotide fingerprinting (ONF) as being differentially expressed. The gene collection contained many unknown genes. Sequence analysis of to date 18.000 cDNA clones led to an estimate of 5.000 non-redundant genes being represented in the collection. 10 % of them are either completely unknown or homologous to rare ESTs (expressed sequence tags) in the public EST database. These clones occurred predominantly in small fingerprint clusters and were therefore assumed to be rarely expressed in DCs or monocytes. Some of those genes may become novel drug targets if their expression is DC specific or induced by external stimuli driving DCs into

  6. Targeting Oxidatively Induced DNA Damage Response in Cancer: Opportunities for Novel Cancer Therapies

    Directory of Open Access Journals (Sweden)

    Pierpaola Davalli

    2018-01-01

    Full Text Available Cancer is a death cause in economically developed countries that results growing also in developing countries. Improved outcome through targeted interventions faces the scarce selectivity of the therapies and the development of resistance to them that compromise the therapeutic effects. Genomic instability is a typical cancer hallmark due to DNA damage by genetic mutations, reactive oxygen and nitrogen species, ionizing radiation, and chemotherapeutic agents. DNA lesions can induce and/or support various diseases, including cancer. The DNA damage response (DDR is a crucial signaling-transduction network that promotes cell cycle arrest or cell death to repair DNA lesions. DDR dysregulation favors tumor growth as downregulated or defective DDR generates genomic instability, while upregulated DDR may confer treatment resistance. Redox homeostasis deeply and capillary affects DDR as ROS activate/inhibit proteins and enzymes integral to DDR both in healthy and cancer cells, although by different routes. DDR regulation through modulating ROS homeostasis is under investigation as anticancer opportunity, also in combination with other treatments since ROS affect DDR differently in the patients during cancer development and treatment. Here, we highlight ROS-sensitive proteins whose regulation in oxidatively induced DDR might allow for selective strategies against cancer that are better tailored to the patients.

  7. Detection of genetically modified organisms (GMOs using isothermal amplification of target DNA sequences

    Directory of Open Access Journals (Sweden)

    La Mura Maurizio

    2009-02-01

    Full Text Available Abstract Background The most common method of GMO detection is based upon the amplification of GMO-specific DNA amplicons using the polymerase chain reaction (PCR. Here we have applied the loop-mediated isothermal amplification (LAMP method to amplify GMO-related DNA sequences, 'internal' commonly-used motifs for controlling transgene expression and event-specific (plant-transgene junctions. Results We have tested the specificity and sensitivity of the technique for use in GMO studies. Results show that detection of 0.01% GMO in equivalent background DNA was possible and dilutions of template suggest that detection from single copies of the template may be possible using LAMP. Conclusion This work shows that GMO detection can be carried out using LAMP for routine screening as well as for specific events detection. Moreover, the sensitivity and ability to amplify targets, even with a high background of DNA, here demonstrated, highlights the advantages of this isothermal amplification when applied for GMO detection.

  8. Label-Free Proteomic Identification of Endogenous, Insulin-Stimulated Interaction Partners of Insulin Receptor Substrate-1

    Science.gov (United States)

    Geetha, Thangiah; Langlais, Paul; Luo, Moulun; Mapes, Rebekka; Lefort, Natalie; Chen, Shu-Chuan; Mandarino, Lawrence J.; Yi, Zhengping

    2011-03-01

    Protein-protein interactions are key to most cellular processes. Tandem mass spectrometry (MS/MS)-based proteomics combined with co-immunoprecipitation (CO-IP) has emerged as a powerful approach for studying protein complexes. However, a majority of systematic proteomics studies on protein-protein interactions involve the use of protein overexpression and/or epitope-tagged bait proteins, which might affect binding stoichiometry and lead to higher false positives. Here, we report an application of a straightforward, label-free CO-IP-MS/MS method, without the use of protein overexpression or protein tags, to the investigation of changes in the abundance of endogenous proteins associated with a bait protein, which is in this case insulin receptor substrate-1 (IRS-1), under basal and insulin stimulated conditions. IRS-1 plays a central role in the insulin signaling cascade. Defects in the protein-protein interactions involving IRS-1 may lead to the development of insulin resistance and type 2 diabetes. HPLC-ESI-MS/MS analyses identified eleven novel endogenous insulin-stimulated IRS-1 interaction partners in L6 myotubes reproducibly, including proteins play an important role in protein dephosphorylation [protein phosphatase 1 regulatory subunit 12A, (PPP1R12A)], muscle contraction and actin cytoskeleton rearrangement, endoplasmic reticulum stress, and protein folding, as well as protein synthesis. This novel application of label-free CO-IP-MS/MS quantification to assess endogenous interaction partners of a specific protein will prove useful for understanding how various cell stimuli regulate insulin signal transduction.

  9. Ultrasensitive electrochemical aptasensor based on sandwich architecture for selective label-free detection of colorectal cancer (CT26) cells.

    Science.gov (United States)

    Hashkavayi, Ayemeh Bagheri; Raoof, Jahan Bakhsh; Ojani, Reza; Kavoosian, Saeid

    2017-06-15

    Colorectal cancer is one of the most common cancers in the world and has no effective treatment. Therefore, development of new methods for early diagnosis is instantly required. Biological recognition probes such as synthetic receptor and aptamer is one of the candidate recognition layers to detect important biomolecules. In this work, an electrochemical aptasensor was developed by fabricating an aptamer-cell-aptamer sandwich architecture on an SBA-15-3-aminopropyltriethoxysilane (SBA-15-pr-NH 2 ) and Au nanoparticles (AuNPs) modified graphite screen printed electrode (GSPE) surface for the selective, label-free detection of CT26 cancer cells. Based on the incubation of the thiolated aptamer with CT26 cells, the electron-transfer resistance of Fe (CN) 6 3-/4- redox couple increased considerably on the aptasensor surface. The results obtained from cyclic voltammetry and electrochemical impedance spectroscopy studies showed that the fabricated aptasensor can specifically identify CT26 cells in the concentration ranges of 10-1.0×10 5 cells/mL and 1.0×10 5 -6.0×10 6 cells/mL, respectively, with a detection limit of 2cells/mL. Applying the thiol terminated aptamer (5TR1) as a recognition layer led to a sensor with high affinity for CT26 cancer cells, compared to control cancer cells of AGS cells, VERO Cells, PC3 cells and SKOV-3 cells. Therefore a simple, rapid, label free, inexpensive, excellent, sensitive and selective electrochemical aptasensor based on sandwich architecture was developed for detection of CT26 Cells. Copyright © 2016 Elsevier B.V. All rights reserved.

  10. Label-free separation of human embryonic stem cells (hESCs) and their cardiac derivatives using Raman spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Chan, J W; Lieu, D K; Huser, T R; Li, R A

    2008-09-08

    Self-renewable, pluripotent human embryonic stem cells (hESCs) can be differentiated into cardiomyocytes (CMs), providing an unlimited source of cells for transplantation therapies. However, unlike certain cell lineages such as hematopoietic cells, CMs lack specific surface markers for convenient identification, physical separation, and enrichment. Identification by immunostaining of cardiac-specific proteins such as troponin requires permeabilization, which renders the cells unviable and non-recoverable. Ectopic expression of a reporter protein under the transcriptional control of a heart-specific promoter for identifying hESC-derived CMs (hESC-CMs) is useful for research but complicates potential clinical applications. The practical detection and removal of undifferentiated hESCs in a graft, which may lead to tumors, is also critical. Here, we demonstrate a non-destructive, label-free optical method based on Raman scattering to interrogate the intrinsic biochemical signatures of individual hESCs and their cardiac derivatives, allowing cells to be identified and classified. By combining the Raman spectroscopic data with multivariate statistical analysis, our results indicate that hESCs, human fetal left ventricular CMs, and hESC-CMs can be identified by their intrinsic biochemical characteristics with an accuracy of 96%, 98% and 66%, respectively. The present study lays the groundwork for developing a systematic and automated method for the non-invasive and label-free sorting of (i) high-quality hESCs for expansion, and (ii) ex vivo CMs (derived from embryonic or adult stem cells) for cell-based heart therapies.

  11. Automated processing of label-free Raman microscope images of macrophage cells with standardized regression for high-throughput analysis.

    Science.gov (United States)

    Milewski, Robert J; Kumagai, Yutaro; Fujita, Katsumasa; Standley, Daron M; Smith, Nicholas I

    2010-11-19

    Macrophages represent the front lines of our immune system; they recognize and engulf pathogens or foreign particles thus initiating the immune response. Imaging macrophages presents unique challenges, as most optical techniques require labeling or staining of the cellular compartments in order to resolve organelles, and such stains or labels have the potential to perturb the cell, particularly in cases where incomplete information exists regarding the precise cellular reaction under observation. Label-free imaging techniques such as Raman microscopy are thus valuable tools for studying the transformations that occur in immune cells upon activation, both on the molecular and organelle levels. Due to extremely low signal levels, however, Raman microscopy requires sophisticated image processing techniques for noise reduction and signal extraction. To date, efficient, automated algorithms for resolving sub-cellular features in noisy, multi-dimensional image sets have not been explored extensively. We show that hybrid z-score normalization and standard regression (Z-LSR) can highlight the spectral differences within the cell and provide image contrast dependent on spectral content. In contrast to typical Raman imaging processing methods using multivariate analysis, such as single value decomposition (SVD), our implementation of the Z-LSR method can operate nearly in real-time. In spite of its computational simplicity, Z-LSR can automatically remove background and bias in the signal, improve the resolution of spatially distributed spectral differences and enable sub-cellular features to be resolved in Raman microscopy images of mouse macrophage cells. Significantly, the Z-LSR processed images automatically exhibited subcellular architectures whereas SVD, in general, requires human assistance in selecting the components of interest. The computational efficiency of Z-LSR enables automated resolution of sub-cellular features in large Raman microscopy data sets without

  12. Label-free reflectance hyperspectral imaging for tumor margin assessment: a pilot study on surgical specimens of cancer patients

    Science.gov (United States)

    Fei, Baowei; Lu, Guolan; Wang, Xu; Zhang, Hongzheng; Little, James V.; Patel, Mihir R.; Griffith, Christopher C.; El-Diery, Mark W.; Chen, Amy Y.

    2017-08-01

    A label-free, hyperspectral imaging (HSI) approach has been proposed for tumor margin assessment. HSI data, i.e., hypercube (x,y,λ), consist of a series of high-resolution images of the same field of view that are acquired at different wavelengths. Every pixel on an HSI image has an optical spectrum. In this pilot clinical study, a pipeline of a machine-learning-based quantification method for HSI data was implemented and evaluated in patient specimens. Spectral features from HSI data were used for the classification of cancer and normal tissue. Surgical tissue specimens were collected from 16 human patients who underwent head and neck (H&N) cancer surgery. HSI, autofluorescence images, and fluorescence images with 2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-D-glucose (2-NBDG) and proflavine were acquired from each specimen. Digitized histologic slides were examined by an H&N pathologist. The HSI and classification method were able to distinguish between cancer and normal tissue from the oral cavity with an average accuracy of 90%±8%, sensitivity of 89%±9%, and specificity of 91%±6%. For tissue specimens from the thyroid, the method achieved an average accuracy of 94%±6%, sensitivity of 94%±6%, and specificity of 95%±6%. HSI outperformed autofluorescence imaging or fluorescence imaging with vital dye (2-NBDG or proflavine). This study demonstrated the feasibility of label-free, HSI for tumor margin assessment in surgical tissue specimens of H&N cancer patients. Further development of the HSI technology is warranted for its application in image-guided surgery.

  13. High-throughput label-free detection of aggregate platelets with optofluidic time-stretch microscopy (Conference Presentation)

    Science.gov (United States)

    Jiang, Yiyue; Lei, Cheng; Yasumoto, Atsushi; Ito, Takuro; Guo, Baoshan; Kobayashi, Hirofumi; Ozeki, Yasuyuki; Yatomi, Yutaka; Goda, Keisuke

    2017-02-01

    According to WHO, approximately 10 million new cases of thrombotic disorders are diagnosed worldwide every year. In the U.S. and Europe, their related diseases kill more people than those from AIDS, prostate cancer, breast cancer and motor vehicle accidents combined. Although thrombotic disorders, especially arterial ones, mainly result from enhanced platelet aggregability in the vascular system, visual detection of platelet aggregates in vivo is not employed in clinical settings. Here we present a high-throughput label-free platelet aggregate detection method, aiming at the diagnosis and monitoring of thrombotic disorders in clinical settings. With optofluidic time-stretch microscopy with a spatial resolution of 780 nm and an ultrahigh linear scanning rate of 75 MHz, it is capable of detecting aggregated platelets in lysed blood which flows through a hydrodynamic-focusing microfluidic device at a high throughput of 10,000 particles/s. With digital image processing and statistical analysis, we are able to distinguish them from single platelets and other blood cells via morphological features. The detection results are compared with results of fluorescence-based detection (which is slow and inaccurate, but established). Our results indicate that the method holds promise for real-time, low-cost, label-free, and minimally invasive detection of platelet aggregates, which is potentially applicable to detection of platelet aggregates in vivo and to the diagnosis and monitoring of thrombotic disorders in clinical settings. This technique, if introduced clinically, may provide important clinical information in addition to that obtained by conventional techniques for thrombotic disorder diagnosis, including ex vivo platelet aggregation tests.

  14. A label-free nanostructured plasmonic biosensor based on Blu-ray discs with integrated microfluidics for sensitive biodetection.

    Science.gov (United States)

    López-Muñoz, Gerardo A; Estevez, M-Carmen; Peláez-Gutierrez, E Cristina; Homs-Corbera, Antoni; García-Hernandez, M Carmen; Imbaud, J Ignacio; Lechuga, Laura M

    2017-10-15

    Nanostructure-based plasmonic biosensors have quickly positioned themselves as interesting candidates for the design of portable optical biosensor platforms considering the potential benefits they can offer in integration, miniaturization, multiplexing, and real-time label-free detection. We have developed a simple integrated nanoplasmonic sensor taking advantage of the periodic nanostructured array of commercial Blu-ray discs. Sensors with two gold film thicknesses (50 and 100nm) were fabricated and optically characterized by varying the oblique-angle of the incident light in optical reflectance measurements. Contrary to the use normal light incidence previously reported with other optical discs, we observed an enhancement in sensitivity and a narrowing of the resonant linewidths as the light incidence angle was increased, which could be related to the generation of Fano resonant modes. The new sensors achieve a figure of merit (FOM) up to 35 RIU -1 and a competitive bulk limit of detection (LOD) of 6.3×10 -6 RIU. These values significantly improve previously reported results obtained with normal light incidence reflectance measurements using similar structures. The sensor has been combined with versatile, simple, ease to-fabricate microfluidics. The integrated chip is only 1cm 2 (including a PDMS flow cell with a 50µm height microfluidic channel fabricated with double-sided adhesive tape) and all the optical components are mounted on a 10cm×10cm portable prototype, illustrating its facile miniaturization, integration and potential portability. Finally, to assess the label-free biosensing capability of the new sensor, we have evaluated the presence of specific antibodies against the GTF2b protein, a tumor-associate antigen (TAA) related to colorectal cancer. We have achieved a LOD in the pM order and have assessed the feasibility of directly measuring biological samples such as human serum. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. An ultrasensitive label-free electrochemiluminescent immunosensor for measuring Cry1Ab level and genetically modified crops content.

    Science.gov (United States)

    Gao, Hongfei; Wen, Luke; Wu, Yuhua; Fu, Zhifeng; Wu, Gang

    2017-11-15

    The development of genetically modified (GM) insect-resistant crops has aroused great public concern about the risks on the eco-environment resulting from a release of toxic Cry proteins (such as Cry1Ab) to the soil. Therefore, it is of crucial importance to measure the Cry proteins level and the GM crops content. Here, we have tested for the first time a method that uses novel carbon nanospheres (CNPs) label-free electrochemiluminescent (ECL) immunosensor for the ultrasensitive quantification of Cry1Ab and GM crops. In this work, novel CNPs were prepared from printer toner with a very facile approach, and linked with anti-Cry1Ab antibodies to modify a golden working electrode. The immunoreaction between Cry1Ab and its antibody formed an immunocomplex on the bioreceptor region of the sensor, which inhibited electron transfer between the electrode surface and the ECL substance, leading to a decrease of ECL response. Under the optimal conditions, the fabricated label-free ECL immunosensor determined Cry1Ab down to 3.0pgmL -1 within a linear range of 0.010-1.0ngmL -1 , showing significant improvement of sensitivity than that of most previous reports. Meanwhile, the proposed method was successfully applied for GM rice BT63 and GM maize MON810 detections down to 0.010% and 0.020%, respectively. Due to its outstanding advantages such as high sensitivity, ideal selectivity, simple fabrication, rapid detection, and low cost, the developed method can be considered as a powerful and pioneering tool for GM crops detection. Its use can also be extended to other toxin protein sensing in foods. Copyright © 2017. Published by Elsevier B.V.

  16. Non-invasive in vivo characterization of skin wound healing using label-free multiphoton microscopy (Conference Presentation)

    Science.gov (United States)

    Jones, Jake D.; Majid, Fariah; Ramser, Hallie; Quinn, Kyle P.

    2017-02-01

    Non-healing ulcerative wounds, such as diabetic foot ulcers, are challenging to diagnose and treat due to their numerous possible etiologies and the variable efficacy of advanced wound care products. Thus, there is a critical need to develop new quantitative biomarkers and diagnostic technologies that are sensitive to wound status in order to guide care. The objective of this study was to evaluate the utility of label-free multiphoton microscopy for characterizing wound healing dynamics in vivo and identifying potential differences in diabetic wounds. We isolated and measured an optical redox ratio of FAD/(NADH+FAD) autofluorescence to provide three-dimensional maps of local cellular metabolism. Using a mouse model of wound healing, in vivo imaging at the wound edge identified a significant decrease in the optical redox ratio of the epidermis (p≤0.0103) between Days 3 through 14 compared to Day 1. This decrease in redox ratio coincided with a decrease in NADH fluorescence lifetime and thickening of the epithelium, collectively suggesting a sensitivity to keratinocyte hyperproliferation. In contrast to normal wounds, we have found that keratinocytes from diabetic wounds remain in a proliferative state at later time points with a lower redox ratio at the wound edge. Microstructural organization and composition was also measured from second harmonic generation imaging of collagen and revealed differences between diabetic and non-diabetic wounds. Our work demonstrates label-free multiphoton microscopy offers potential to provide non-invasive structural and functional biomarkers associated with different stages of skin wound healing, which may be used to detect delayed healing and guide treatment.

  17. "Peak tracking chip" for label-free optical detection of bio-molecular interaction and bulk sensing.

    Science.gov (United States)

    Bougot-Robin, Kristelle; Li, Shunbo; Zhang, Yinghua; Hsing, I-Ming; Benisty, Henri; Wen, Weijia

    2012-10-21

    A novel imaging method for bulk refractive index sensing or label-free bio-molecular interaction sensing is presented. This method is based on specially designed "Peak tracking chip" (PTC) involving "tracks" of adjacent resonant waveguide gratings (RWG) "micropads" with slowly evolving resonance position. Using a simple camera the spatial information robustly retrieves the diffraction efficiency, which in turn transduces either the refractive index of the liquids on the tracks or the effective thickness of an immobilized biological layer. Our intrinsically multiplex chip combines tunability and versatility advantages of dielectric guided wave biochips without the need of costly hyperspectral instrumentation. The current success of surface plasmon imaging techniques suggests that our chip proposal could leverage an untapped potential to routinely extend such techniques in a convenient and sturdy optical configuration toward, for instance for large analytes detection. PTC design and fabrication are discussed with challenging process to control micropads properties by varying their period (step of 2 nm) or their duty cycle through the groove width (steps of 4 nm). Through monochromatic imaging of our PTC, we present experimental demonstration of bulk index sensing on the range [1.33-1.47] and of surface biomolecule detection of molecular weight 30 kDa in aqueous solution using different surface densities. A sensitivity of the order of 10(-5) RIU for bulk detection and a sensitivity of the order of ∼10 pg mm(-2) for label-free surface detection are expected, therefore opening a large range of application of our chip based imaging technique. Exploiting and chip design, we expect as well our chip to open new direction for multispectral studies through imaging.

  18. Epsilon-Q: An Automated Analyzer Interface for Mass Spectral Library Search and Label-Free Protein Quantification.

    Science.gov (United States)

    Cho, Jin-Young; Lee, Hyoung-Joo; Jeong, Seul-Ki; Paik, Young-Ki

    2017-12-01

    Mass spectrometry (MS) is a widely used proteome analysis tool for biomedical science. In an MS-based bottom-up proteomic approach to protein identification, sequence database (DB) searching has been routinely used because of its simplicity and convenience. However, searching a sequence DB with multiple variable modification options can increase processing time, false-positive errors in large and complicated MS data sets. Spectral library searching is an alternative solution, avoiding the limitations of sequence DB searching and allowing the detection of more peptides with high sensitivity. Unfortunately, this technique has less proteome coverage, resulting in limitations in the detection of novel and whole peptide sequences in biological samples. To solve these problems, we previously developed the "Combo-Spec Search" method, which uses manually multiple references and simulated spectral library searching to analyze whole proteomes in a biological sample. In this study, we have developed a new analytical interface tool called "Epsilon-Q" to enhance the functions of both the Combo-Spec Search method and label-free protein quantification. Epsilon-Q performs automatically multiple spectral library searching, class-specific false-discovery rate control, and result integration. It has a user-friendly graphical interface and demonstrates good performance in identifying and quantifying proteins by supporting standard MS data formats and spectrum-to-spectrum matching powered by SpectraST. Furthermore, when the Epsilon-Q interface is combined with the Combo-Spec search method, called the Epsilon-Q system, it shows a synergistic function by outperforming other sequence DB search engines for identifying and quantifying low-abundance proteins in biological samples. The Epsilon-Q system can be a versatile tool for comparative proteome analysis based on multiple spectral libraries and label-free quantification.

  19. [Identification of Clonorchis sinensis metacercariae based on PCR targeting ribosomal DNA ITS regions and COX1 gene].

    Science.gov (United States)

    Yang, Qing-Li; Shen, Ji-Qing; Jiang, Zhi-Hua; Yang, Yi-Chao; Li, Hong-Mei; Chen, Ying-Dan; Zhou, Xiao-Nong

    2014-06-01

    To identify Clonorchis sinensis metacercariae using PCR targeting ribosomal DNA ITS region and COX1 gene. Pseudorasbora parva were collected from Hengxian County of Guangxi at the end of May 2013. Single metacercaria of C. sinensis and other trematodes were separated from muscle tissue of P. parva by digestion method. Primers targeting ribosomal DNA ITS region and COX1 gene of C. sinensis were designed for PCR and the universal primers were used as control. The sensitivity and specificity of the PCR detection were analyzed. C. sinensis metacercariae at different stages were identified by PCR. DNA from single C. sinensis metacercaria was detected by PCR targeting ribosomal DNA ITS region and COX1 gene. The specific amplicans have sizes of 437/549, 156/249 and 195/166 bp, respectively. The ratio of the two positive numbers in PCR with universal primers and specific primers targeting C. sinensis ribosomal DNA ITS1 and ITS2 regions was 0.905 and 0.952, respectively. The target gene fragments were amplified by PCR using COX1 gene-specific primers. The PCR with specific primers did not show any non-specific amplification. However, the PCR with universal primers targeting ribosomal DNA ITS regions performed serious non-specific amplification. C. sinensis metacercariae at different stages are identified by morphological observation and PCR method. Species-specific primers targeting ribosomal DNA ITS region show higher sensitivity and specificity than the universal primers. PCR targeting COX1 gene shows similar sensitivity and specificity to PCR with specific primers targeting ribosomal DNA ITS regions.

  20. Impedimetric DNA Biosensor Based on a Nanoporous Alumina Membrane for the Detection of the Specific Oligonucleotide Sequence of Dengue Virus

    Directory of Open Access Journals (Sweden)

    Chee-Seng Toh

    2013-06-01

    Full Text Available A novel and integrated membrane sensing platform for DNA detection is developed based on an anodic aluminum oxide (AAO membrane. Platinum electrodes (~50–100 nm thick are coated directly on both sides of the alumina membrane to eliminate the solution resistance outside the nanopores. The electrochemical impedance technique is employed to monitor the impedance changes within the nanopores upon DNA binding. Pore resistance (Rp linearly increases in response towards the increasing concentration of the target DNA in the range of 1 × 10−12 to 1 × 10−6 M. Moreover, the biosensor selectively differentiates the complementary sequence from single base mismatched (MM-1 strands and non-complementary strands. This study reveals a simple, selective and sensitive method to fabricate a label-free DNA biosensor.

  1. Impedimetric DNA biosensor based on a nanoporous alumina membrane for the detection of the specific oligonucleotide sequence of dengue virus.

    Science.gov (United States)

    Deng, Jiajia; Toh, Chee-Seng

    2013-06-17

    A novel and integrated membrane sensing platform for DNA detection is developed based on an anodic aluminum oxide (AAO) membrane. Platinum electrodes (~50-100 nm thick) are coated directly on both sides of the alumina membrane to eliminate the solution resistance outside the nanopores. The electrochemical impedance technique is employed to monito